Showing papers in "Plant Molecular Biology in 1985"

Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues

Abstract: We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.

Pathogenesis-related proteins.

Abstract: Behandeling van het optreden en eigenschappen van - in verband met het ontstaan van planteziekten voorkomende - eiwitten, de inductie en mogelijke functies van deze eiwitten

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding.

Abstract: Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Abstract: We show here that plant cells are sensitive to the antibiotic hygromycin-B4. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.

Resistance to hygromycin B : A new marker for plant transformation studies.

Abstract: A bacterial gene encoding hygromycin phosphotransferase has been modified for expression in tobacco cells. The aphIV gene from Escherichia coli was inserted between the 5′ sequence of an octopine synthase gene and the 3′ sequence from a nopaline synthase gene. The new gene was incorporated between T-DNA border fragments in the broad-host-range vector pKT210 to form a micro-Ti plasmid. Agrobacterium tumefaciens containing this plasmid and a Ti plasmid as helper was used to incite crown gall tumors on aseptic tobacco plants. Samples of these galls could grow in the presence of hygromycin B, provided that the aph gene had been fused with the ocs gene to maintain the sense of the coding sequences. When the genes had been fused in the reverse ‘antisense’ orientation none of the gall samples could grow on hygromycin. Unlike wild-type galls the hygromycin-resistant tissue contained DNA sequences homologous to the aphIV gene. Thus the modified gene can be introduced into tobacco cells and confer on them the ability to grow in the presence of hygromycin B.

Isolation and characterisation of cDNA clones for tomato polygalacturonase and other ripening-related proteins.

Abstract: Gene expression during the ripening of tomato fruit was investigated by cDNA cloning and hybrid-select translation. A cDNA library was prepared from poly(A)-containing mRNA from ripe tomato fruit and sreened by differential hybridization. 146 ripening-related cDNA clones were found. Eleven groups and eight unique clones have been identified so far. The sizes of the cloned cDNA inserts were determined and type-members for seven groups were used in hybrid selection experiments. Six of the seven clones encode translation products corresponding to six ripening related polypeptides detected previously by in vitro translation of total cytoplasmic RNA (14). One cDNA group codes for a Mr 48 000 protein that was identified as polygalacturonase on the basis of immunoprecipitation with specific antiserum raised against tomato polygalacturonase. re]19840918 rv]19850613 ac]19850618

Nucleotide sequence of the 17S-25S spacer region from rice rDNA.

Abstract: The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194-195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163-164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5' terminal region of 25S rDNA.

Multiple transcripts for higher plantrbcL andatpB genes and localization of the transcription initiation site of therbcL gene.

Abstract: We have compared therbcL andatpB transcription units from spinach, maize, and pea. In most cases multiple transcripts were found for a given chloroplast gene. The 5′ termini of these transcripts were determined by S1 nuclease protection and primer extension analyses. TherbcL transcripts have 5′ termini 178–179 and 64 nucleotides (spinach), 300 and 59–63 nucleotides (maize), and 178 and 65 nucleotides (pea) upstream from their respective protein coding regions. TheatpB transcripts have 5′ termini (453–454, 272–273, 179, and 99 nucleotides (spinach), 298–302 nucleotides (maize), and 351–355 nucleotides (pea) upstream from their respective protein coding regions. The intergenic distance between therbcL andatpB genes is relatively constant (152 to 157 base pairs) among the three chloroplast genomes. In spinach, maize, and pea, the 80 base pairs surrounding the 5′ end of therbcL gene (±40 base pairs) have 85% sequence homology. Similarly, the 60 base pairs preceding theatpB gene have 48% sequence homology. Both genes have ‘−10’ and ‘−35’ regions that resemble the prokaryotic consensus promoter sequence. The larger, but not smaller,rbcL transcripts from spinach and pea can be labeled with alpha-32P-GTP by guanylyltransferase. These data suggest that DNA sequences 178–179 (spinach), 300 (maize), and 178 (pea) base pairs before therbcL protein coding regions represent sites of transcription initiation. The sequences 59–65 base pairs before therbcL protein coding regions may correspond to sites of RNA cleavage.

Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum

Abstract: The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with 14C-leucine, we have shown that the 30 kd α polypeptides and 20 kd β polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the α polypeptide precedes the β polypeptide on the precursor. Cruciferin and legumin share 40% homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the α subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the α subunit, which is not found in cruciferin. Both of these regions appear to have originated by reiteration of sequences. re]19850513 ac]19850715

Comparison of multimeric plus and minus forms of viroids and virusoids

Abstract: In order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication.The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series.The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids.

Abstract: We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped.Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.

Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855

Abstract: Root proliferation can be induced by Agrobacterium rhizogenes on carrot discs both on the apical and basal surface (facing the root apex and base, respectively) or on the apical surface only, depending on the bacterial strain. This differential response on the two surfaces is denominated polarity. We correlate the polarity of some strains with the absence of an Ri plasmid genetic locus, present in non polar strains such as A. rhizogenes 1855, which bears sequence homology with the auxin genes of Ti plasmid T-DNA. We demonstrate that this locus is responsible for root induction on the basal surface since insertion of a transposon in this region of pRi1855 induces polarity in this strain.

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex.

Abstract: Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984). Poly(A)+RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)+RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells. Dot-blot and northern-blot hybridizations of poly(A)+RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)+RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)+RNA. Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613

Simple binary vectors for DNA transfer to plant cells

Abstract: Cosmid binary vectors for the introduction of DNA into plant cells have been constructed. These vectors are derived from the replicon of the broad host range plasmid pRK2 and contain the T-DNA border regions between which have been placed a chimaeric gene conferring resistance to kanamycin in plant cells. Appropriate restriction endonuclease targets have also been placed between the border regions. These binary vectors, in conjunction with appropriate Agrobacterium strains, are capable of delivering DNA to plant cells in cocultivation experiments with very high efficiency. The transformation frequency is shown to be somewhat dependent on the replicon used. re]19850121 rv]19850506 ac]19850513

α-amylase genes of wheat are two multigene families which are differentially expressed.

Abstract: The α-Amy1 and α-Amy2 genes of wheat produce distinct subsets of α-amylase isozymes which show different patterns of expression in wheat aleurone cells and in developing grain. In order to characterise the organisation and expression of these genes, clones of α-Amy1 and α-Amy2 cDNA have been isolated. The two types of cDNA clone were distinguished within a small library of α-amylase cDNA clones (Baulcombe and Buffard, Planta 157 493–501 [1983]) by restriction endonuclease mapping and by cross hybridisation. The identity of α-Amy1 or α-Amy2 type was assigned from the results of hybrid selected translation analysis in which small subfragments of the cDNA clones were used. These subfragments were derived from the 3′ ends of the cDNA and did not cross hybridise between the different types of cDNA. Hybridisation of α-Amy1 and α-Amy2 cDNA probes to restriction enzyme digests of wheat nuclear DNA revealed that these are multigene families located on the group 6 (α-Amy1) and group 7 (α-Amy2) chromosomes. Studies on the levels of α-Amy1 and α-Amy2 mRNA in developing grain and in aleurone tissue indicated that the differences in isozyme expression are due to the patterns of mRNA accumulation. In aleurone tissue the α-Amy1 transcripts accumulate in parallel with other genes which are regulated by gibberellic acid, while the accumulation of α-Amy2 genes is sustained for 36 h longer.

Phytochrome regulation of phytochrome mRNA abundance

Abstract: Pure phytochrome RNA sequence synthesized in an SP6-derived in vitro transcription system has been used as a standard to quantitate phytochrome mRNA abundance in Avena seedlings using a filter hybridization assay. In 4-day-old etiolated Avena seedlings phytochrome mRNA represents ∼0.1% of the total poly(A)(+) RNA. Irradiation of such seedlings with a saturating red-light pulse or continuous white light induces a decline in this mRNA that is detectable within 30 min and results in a 50% reduction by ∼60 min and >90% reduction within 5 h. The effect of the red-light pulse is reversed, approximately to the level of the far-red control, by an immediately subsequent far-red pulse. In seedlings maintained in extended darkness after the red-light pulse, the initial rapid decline in phytochrome mRNA level is followed by a slower reaccumulation such that 50-60% of the initial abundance is reached by 48 h. White-light grown seedlings transferred to darkness exhibit a similar accumulation of phytochrome mRNA that is accelerated by removal of residual Pfr with a far-red light pulse at the start of the dark period. The data establish that previously reported phytochrome-regulated changes in translatable phytochrome mRNA levels result from changes in the physical abundance of this mRNA rather than from altered translatability.

Plastid gene expression during fruit ripening in tomato

Abstract: A tomato chloroplast genome map has been constructed with the restriction enzymes Hpa I, Pvu II, and Sal I. Twelve plastid genes have been located on the tomato plastid genome (159 kb).

Nucleotide sequences of the genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase.

Abstract: The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.

Intracellular coding sites of polypeptides associated with photosynthetic oxygen evolution of photosystem II.

Abstract: Three hydrophilic polypeptides of approximately 34, 23, and 16 kd located on the inner thylakoid surface are associated with the water-splitting activity of photosystem II. Stable transcripts for the three proteins were found only in cytosolic (polyadenylated) RNA, suggesting that they are encoded in nuclear genes. The immunologically reacting products synthesized in a rabbit reticulocyte cell-free translation system are larger in size than the authentic mature proteins by about 6–10 kd. These larger precursors are imported post-translationally into isolated, intact chloroplasts, and are processed to their mature forms during or after translocation. The imported proteins can be extracted from thylakoids by procedures used to isolate the three native proteins of the water-splitting complex, suggesting that they have assembled properly into their final destination, the inner thylakoid surface.

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved.

Abstract: Five specific transposon-induced nodulation defective (Nod−) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod− mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod− on all their known plant hosts, (b), they could not induce root hair curling (Hac−) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.

Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots.

Abstract: Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)+ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.

Cellular double-stranded RNA in Phaseolus vulgaris.

Abstract: High molecular weight double-stranded (ds) RNAs have been detected in apparently virus-free French (common) bean Phaseolus vulgaris cv. Black Turtle Soup (BTS). Several other bean cultivars were free of detectable high molecular weight dsRNAs. The dsRNAs have been partially characterized and have homology to the BTS genome as well as to the genomes of other bean cultivars. The Tm of hybrids formed between BTS DNA and denatured dsRNA have been estimated.

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Abstract: Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N2-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N2 fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N2-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.

Biogenesis of photosystem I reaction center during greening of oat, bean and spinach leaves.

Abstract: The relative amounts of some chloroplast polypeptides were followed during greening of leaves from three different plant families. Oat, bean and spinach were the representatives of the Gramineae, Leguminosae and Chenopodiaceae, respectively. By using specific antibodies against subunits of the chloroplast protein complexes, it was found with that method that the protein complexes which are not involved in a photobiochemical reaction were synthesized in etiolated leaves and their amounts did not significantly change during greening. Examples of these are the large and small subunits of ribulose 1,5-bisphosphate carboxylase, the subunits of the chloroplast coupling factor (CF1) and cytochrome b6-f complex. On the other hand, in photosystem I reaction center, the synthesis of subunits II, III, IV and V was found to be induced by light. Sequential synthesis of these subunits was observed. Subunit II is the first to be synthesized after exposing the plants to light. The synthesis of subunits III, IV and V followed the synthesis of subunit II in this order. Subunit I of photosystem I reaction center was present in etiolated leaves and its amount was not significantly altered during the first few hours of greening.

The disease resistance response in pea is associated with increased levels of specific mRNAs.

Abstract: A cDNA library was constructed using poly(A)+RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using32P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of3H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.

Plant-virus-based vectors for gene transfer will be of limited use because of the high error frequency during viral RNA synthesis

Abstract: The error frequency during the RNA replication of alfalfa mosaic virus (AMV) was calculated to be significantly higher than 10−5. It may be expected that RNA synthesis in general will have low fidelity compared to DNA synthesis. The low fidelity of RNA replication will severely restrict the usefulness of vectors for genetic engineering which are based on RNA viruses, viroids or DNA viruses which are replicated via an RNA intermediate (e.g. caulimoviruses). Spontaneous mutants selected by host shift were found to be much less stable than UV-induced mutants. This difference points to variations in fidelity during RNA synthesis, probably due to the local sequence of the template.

The detection of PR (b) protein and TMV by ELISA in systemic and localised virus infections of tobacco

Abstract: An antiserum was prepared to the b1 protein purified from TMV infectedN. tabacum cv. Xanthi-nc leaves and used to study PR proteins. The Xanthi-nc proteins b2 and b3 were shown to be serologically closely related to b1. Antisera to b1 protein and TMV were used in a F(ab′)2 enzyme linked immunosorbent assay to monitor PR protein and TMV concentrations, respectively, during the first 6 days of a systemic TMV infection (cv. Xanthi) and a localised TMV infection (cv. Xanthi-nc).

T-DNA genes to study plant development: precocious tuberisation and enhanced cytokinins in A. tumefaciens transformed potato

Abstract: Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100–200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3–10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.

Localization of the gene for apocytochromeb-559 on the plastid chromosome of spinach.

Abstract: The gene for cytochromeb-559, associated with the photosystem II reaction center, has been located on the spinach plastid chromosome by cell-free coupled transcription-translation and RNA-programmed hybrid selection translation using appropriate recombinant DNAs, RNA fractions, and monospecific antisera. The gene is located in the large single-copy segment of the plastid chromosome between the genes for cytochomef and the P680 chlorophylla apoprotein of photosystem II and transcribed in the opposite direction relative to these genes. The 10 kd protein is decoded from a bicistronic 1.0 kb mRNA and is apparently not made as a precursor in cell-free rabbit reticulocyte andE. coli lysates.

Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Abstract: With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T+), also had acquired non-selected calf thymus carrier DNA sequences (C+), being integrated in their nuclear genomes From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas+), shoots were grafted and mature, flowering plants (gNT4) were obtained After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features The mannopine locus segregated independently from the locus determining the crown gall phenotype When screened for integrated (‘transforming’) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C+T+ Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C+T+:C-T+: C+T:C T SR1-like seedlings Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes