Showing papers in "Plant Molecular Biology in 1987"

Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Abstract: A region of 16.8 kb of the Sym(biosis) plasmid pRL1JI of Rhizobium leguminosarum, consisting of the established 9.7 kb nodulation region which confers nodulation ability on Vicia hirsuta and a region of 7.1 kb which appeared to be necessary for nodulation on V. sativa and Trifolium subterraneum, was subcloned as fragments of maximally 2.5 kb in a newly developed IncQ transcriptional fusion vector. The expression of these fragments was studied in Rhizobium. One constitutive promoter, pr.nodD, and three plant-exudate inducible promoters were found, namely the known pr.nodA and pr.nodF as well as a new promoter designated pr.nodM. The latter promoters were localized within 114 bp, 330 bp and 630 bp respectively and they regulate the transcription of the operons nodA, B, C, I, J, nodF, E and of an operon of at least 2.5 kb located in the 7.1 kb region. Induction of the three inducible operons required plant exudate and a functional nodD product. The flavanone naringenin could replace plant exudate. Each of the three inducible promoters contained a nod-box. A consensus for the nod-box sequence, based on known sequences, is proposed. The 114 bp fragment which contains pr.nodA activity was used to localize pr.nodA by means of deletion mapping. The fragment which appeared necessary for complete pr.nodA activity is 72 bp in size, contains the complete nod-box and in addition a region of 21 bp downstream of the nod-box, in which the loosely conserved sequence AT(T)AG appears to be important for promoter activity.

Hybrid genes in the analysis of transformation conditions : I. Setting up a simple method for direct gene transfer in plant protoplasts.

Abstract: Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR1 tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR1 Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl2 and PEG. The technique yielded up to 1400 transformants per 3×105 treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the ‘competence’ for transformation has a species-specific component.

Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer.

Abstract: Ribosomal RNA genes in plants are highly variable both in copy number and in intergenic spacer (IGS) length. This variability exists not only between distantly related species, but among members of the same genus and also among members of the same population of a single species. Analysis of inheritance indicates that copy number change is rapid, occurring even among somatic cells of individual plants, and that up to 90% or more of the gene copies are superfluous. Subrepetitive sequences within the IGS appear to be changing rapidly as well. They are not only variable in sequence from one species to the next, but can vary in number between neighboring gene repeats on the chromosome. In all species examined in detail they are located in the same region of the IGS and contain sequences that can be folded into stem-loop structures flanked by a pyrimidine-rich region. It has been suggested that these subrepeats function in transcriptional enhancement, termination or processing, or in recombination events generating the high multiplicity of ribosomal genes.

Plant gene expression in response to pathogens.

Abstract: Introduction 389 A note on terminology 389 Gene expression in plant defence responses 392 (1) The targeted approach 392 (2) The shotgun approach 392 Defence responses involving secondary plant metabolism 394 (1) Phytoalexins 394 (a) Parsley (Petrose/inum crispum) 394 (b) French bean (Phaseolus vulgaris) 395 (c) Soybean (Glycine max) 396 (d) Castor bean (Ricinus communis) 396 (2) Lignification Plant cell wall modification other than lignification Hydrolytic enzymes (1) Chitinase (2) {3-1,3-glucanase Pathogenesis-related proteins Proteinase (protease) inhibitors Other proteins The hypersensitive reaction (HR) Conclusions Acknowledgments References 396 397 398 398 398 398 399 399 399 401 403 403

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants.

Abstract: Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs.

Abstract: Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 μM 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens.

Abstract: High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 μg/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.

Identification, purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves.

Abstract: Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.

Segregation of genes transferred to one plant cell from two separate Agrobacterium strains.

Abstract: Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.

Structure and variation in ribosomal RNA genes of pea : Characterization of a cloned rDNA repeat and chromosomal rDNA variants.

Abstract: A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic ‘nontranscribed spacer’ (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.

Sequencing and modification of psbB, the gene encoding the CP-47 protein of Photosystem II, in the cyanobacterium Synechocystis 6803.

Abstract: The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.

Paternal inheritance of chloroplast DNA in Larix.

Abstract: Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine.

Abstract: The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology, chromosomal localization and evolutionary aspects

Abstract: Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.

The globulin seed storage proteins of flowering plants are derived from two ancestral genes

Abstract: The cDNA and/or genomic DNA sequences of 13 globulin storage proteins from flowering plants (angiosperms) are now known. They represent 8 genera, 5 families and 5 orders of plants and include one monocotyledonous species. Here, the coding nucleotide and amino acid sequences of these proteins are compared by dot matrix analysis and gross protein domains visualized by hydropathy analyses. The vestigial homologies visualized by these means indicate that all of the globulin storage proteins of flowering plants have emanated from 2 genes that existed at the beginning of angiosperm evolution.A curious polypeptide domain of 150-200 amino acids located near the N terminus is found in a globulin subgroup of 2 genera widely separated phylogenetically. The domain appears to have resulted from an ancient insertion that has been deleted in most of its descendant genes.

urf13-T of T Cytoplasm Maize Mitochondria Encodes a 13 kD Polypeptide

Abstract: Polyspecific antibody to a 17 amino acid synthetic peptide from the maize T-cytoplasm urf13-T mitochondrial open reading frame immunoprecipitated a 13 kD polypeptide from (35)S-methionine incorporations of T cytoplasm maize. Male-fertile, toxin-insensitive mutants in which the urf13-T sequence is deleted do not synthesize the 13 kD polypeptide. A mutant designated T-4, which carries a 5 bp insertion and a premature stop codon, synthesizes a truncated polypeptide, corresponding to an open reading frame of 8.3 kD. Thus the 13 kD polypeptide is trunctated or absent in mutants expressing male fertility and toxin insensitivity in T-cytoplasm maize.

Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa)

Abstract: Expression of plant genes involved in the symbiosis between alfalfa (Medicago sativa) and Rhizobium meliloti has been studied by comparing root and root nodule mRNA populations. Two-dimensional gel electrophoretic separation of the in vitro translation products of polyA+ RNA isolated from either roots or effective root nodules has allowed us to identify thirteen nodule-specific translation products, including those corresponding to the leghemoglobins (Lb). These translation products, representing putative nodulin mRNAs, are first detected between 9 and 12 days after inoculation, a result which has been confirmed for Lb mRNA by Northern blotting and hybridization with a Lb cDNA probe. Analysis of three different types of ineffective root nodules arrested in different stages of development has led to the following conclusions. (i) The transcription of eleven nodule-specific genes, including the Lb genes, is independent of nitrogen-fixing activity. (ii) Differentiation of the primary nodule structure does not require the transcription of any of these genes but can be correlated with a dramatic reduction in the level of at least five transcripts present in the root. (iii) There is enhanced expression of certain plant genes in the case of nodules elicited by an Agrobacterium strain carrying the symbiotic plasmid of R. meliloti.

Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: effect of light and acetate

Abstract: In Chlamydomonas reinhardtii, the chlorophyll a/b-binding proteins of photosystem II are encoded in the nucleus by a small family of genes. We have studied the expression of one gene, which we call cabII-1, in a green-in-the-dark strain, which can synthesize chlorophyll in the dark or light, and in a yellow-in-the-dark mutant strain, which is able to make chlorophyll only in the light. In light/dark synchronized cultures of both strains, cabII-1 mRNA abundance increases during the first 6 h of a 12-h light phase, remains high for several hours, then declines. A variety of illumination conditions have been used to analyze the cabII-1 mRNA increase: continuous or intermittent red, blue, or white light, with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Our results suggest that light induces increased cabII-1 transcript abundance in two ways: 1) by virtue of its role in the light reactions of photosynthesis and 2) by a blue lightstimulated mechanism which is independent of photosynthesis.

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002.

Abstract: The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5' to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25-26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome.

Abstract: The aurea locus mutant (au (w)) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158-165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.

The tomato Cab-4 and Cab-5 genes encode a second type of CAB polypeptides localized in Photosystem II.

Abstract: The photosynthetic apparatus of plant chloroplasts contains two photosystems, termed Photosystem I (PSI) and Photosystem II (PSII). Both PSI and PSII contain several types of chlorophyll a/b-binding (CAB) polypeptides, at least some of which are structurally related. It has been previously shown that multiple genes encoding one type of PSII CAB polypeptides exist in the genome of many higher plants. In tomato, there are at least eight such genes, distributed in three independent loci. Genes encoding a second type of CAB polypeptides have been isolated from several plant species, but the precise location of the gene products has not been determined. Here we show that tomato has two unlinked genes encoding this second type and that this type of CAB polypeptide is also localized in PSII.

Identification of psbA and psbD gene products, D1 and D2, as reaction centre proteins of photosystem 2

Abstract: A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109-112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.

Auxin-induced mRNA species in tobacco cell cultures.

Abstract: Using a 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell-suspension culture we analyzed early hormone-induced molecular events preceding cell division. By differential screening of a cDNA library to mRNAs derived from hormone-starved cells treated with 2,4-D for 4 h, seven non-cross hybridizing cDNA clones to 2,4-D-induced mRNAs were obtained. Accumulation of these mRNAs started as early as 15–30 min or less after 2,4-D application. The lowest 2,4-D concentration necessary to induce the mRNAs varied between less than 2.2 × 10−8 M and 2.2 × 10−6 M, one mRNA being induced to nearly maximal values at 2.2 × 10−6 M. Generally, 2,4-D was the most active compound to induce mRNA accumulation, followed by naphthalene-1-acetic acid (NAA). The level of 4 mRNAs increased independently from protein synthesis. Run-off transcription studies showed that the accumulation of some mRNAs was at least partly due to enhanced transcription rates. In different organs of the tobacco plant, the levels of the mRNAs were about as low as in hormone-starved cells. A similar low level of the 2,4-D-induced mRNAs was observed in cells growing in mid-log phase on 2,4-D-containing medium. Only quiescent cells that were triggered to undergo cell division, accumulate these mRNAs transiently.

Expression of a soybean β-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues

Abstract: A gene encoding the α′-subunit of β-conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of α′-polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the α′-polypeptide reflect enhanced stability of this protein.

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata.

Abstract: Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to “nod-box”-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Abstract: Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.

Structure and organization of two divergent α-amylase genes from barley.

Abstract: We have isolated several α-amylase genomic clones from an Eco RI library of barley DNA in λ-Charon 32. Five of these clones exhibit unique restriction maps and differences in their abilities to hybridize with two previously characterized α-amylase cDNA probes representing two different loci, α-Amy 1 (high pI) and α-Amy 2 (low pI) on barley chromosomes 6 and 1, respectively. Stringent hybridizations indicate that four of the five genomic clones contain α-Amy 1 sequences and one contains α-Amy 2 sequences. The regions containing α-amylase genes from one representative genomic clone of each group have been sub-cloned, mapped and sequenced. S1-nuclease protection experiments indicate that the two α-amylase genes contained in these clones are functional in aleurone tissue. Transcription start sites in these genes were determined by primer extension using specific synthetic oligonucleotide primers.The DNA sequences of the two α-amylase genes, including promoter regions, are divergent, as are the predicted amino acid sequences of the mature proteins and the N-terminal "leader" peptides. The α-Amy 1 gene contains two introns while the α-Amy 2 gene has three introns. In the coding region, each gene shows 7-10% sequence divergence with respect to the previously characterized cDNA clones of the same gene type. Therefore, differences in nucleotide sequences can account for some of the isozyme variations seen between the sub-families of α-amylases and among members of the same subfamily. Although the nucleotide sequences of the promoter regions of α-Amy 1 and α-Amy 2 genes show little homology, both contain pairs of inverted repeat elements which could constitute regulatory sites.

Molecular characterization of a wound-inducible inhibitor I gene from potato and the processing of its mRNA and protein.

Abstract: Genomic blotting of restriction fragments of Russet Burbank DNA indicated that at least 6 copies of Inhibitor I are present in the tetraploid potato genome. A library of potato genes in bacteriophage λ was screened for the presence of Inhibitor I genes using a wound-inducible tomato Inhibitor I cDNA as a hybridization probe. One phage with an insert of 13.1 kb was isolated that hybridized most strongly with the probe. A 4.2 kb Eco RI fragment containing the gene was isolated from the clone and 2.2 kb region was sequenced that included about 800 bp of both the 5′ and 3′ regions. The gene contained two introns of 479 and 417 bp respectively, and the splice junctions were typical of other eukaryotic genes. Putative TATAA and CAAT boxes were identified. The nucleotide sequence, when compared with a wound-inducible tomato Inhibitor I cDNA, exhibited over 90% identity. The gene codes for a prepro-Inhibitor I protein of 96 amino acids. The putative pre-sequence of 19 amino acids, differs in only one residue from that of tomato Inhibitor I. The potato pro-sequence, however, is lacking a tetrapeptide that is found in the tomato pro-sequence in the region of pro-peptide processing. This deletion, together with a substitution of a Gln for a Leu (4 residues toward the N terminus) provides an explanation for the differences at the N-termini between tomato and potato Inhibitor I natural proteins by providing different processing sites in the two pro-inhibitors. Thus, amino acid sequence differences between the N termini of tomato and potato Inhibitor I are easily explained by the mutational events. The different proposed pro-processing sites of the tomato and potato inhibitors suggest that a processing protease may be present in the vacuole with a specificity for Asn-X and Gln-X bonds.

The relatively large beta-tubulin gene family of Arabidopsis contains a member with an unusual transcribed 5' noncoding sequence

Abstract: We have characterized the beta-tubulin gene family of Arabidopsis thaliana. Five distinct genes were cloned and analyzed by restriction enzyme mapping and cross-hybridization studies. Three of the genes appear to be dispersed, whereas two others are linked within 1.5 kb of one another. The two linked genes are closely related and appear to have resulted from a fairly recent duplication. The three dispersed genes do not cross-hybridize to one another or to the two linked genes under highly stringent hybridization conditions, suggesting that they arose from more ancient duplications. From Southern analysis we estimate that there are a total of between six and ten beta-tubulin genes in Arabidopsis. Additional analyses indicate that the gene family is equal in size or larger than those in other plants, but significantly smaller than those in related Brassica species. Sequence determination of one of the Arabidopsis genes revealed a highly unusual transcribed leader sequence. The leader contains two fairly long tracks of adenines. One is located toward the 5′ end of the mRNA and the other is just before the initiation codon. A track of uridines is located between the adenine tracks. This leader can form two different secondary structures that may have regulatory significance.

Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin.

Abstract: We have examined the effects of novobiocin and template topology on the transcription of two chloroplast genes encoding the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the chloroplast ATPase (atpB), in an in vitro transcription system. The template topology was monitored by agarose gel electrophoresis while the in vitro transcripts were determined by 5′ S1 nuclease analysis under identical conditions. We discovered that our chloroplast transcription extracts contain a DNA gyrase activity and a chromatographically separable topoisomerase I activity. Incubation of a supercoiled template with the extracts under the same conditions in which transcription assays were carried out leads to a decrease in the supercoiled from and concomitant appearance of distinct topoisomers. More extensive relaxation of the supercoiled template occurs when nucleotide triphosphates are omitted from the reaction mixture or when a low concentration (25 μg/ml) of novobiocin is added. Higher concentrations (≥ 250 μg/ml) of the drug, however, also inhibit the topoisomerase I activity. The transcription of the atpB gene is inhibited by lower concentrations of novobiocin as compared to the rbcL gene in the same reaction mixture. Relaxed, closed circular template and linearized DNA are not substrates for chloroplast transcription extracts, although they are transcribed accurately by the E. coli RNA polymerase under our conditions. Control of in vitro transcription of the two chloroplast genes by template topology can also be demonstrated by modulating the relative activity for the topoisomerases in the transcription extract. Our results suggest that changes in template topology may be a mechanism by which chloroplast genes are differentially regulated and the chloroplast DNA gyrase and topoisomerase I are key enzymes for this mode of regulation in vivo.