Showing papers in "Plant Molecular Biology in 1982"

Identification of T-DNA in the root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855.

Abstract: The region of the virulence plasmid of the agropine type Agrobacterium rhizogenes 1855 (pRi-1855) which is transferred to plant cells upon infection (T-DNA) has been identified by means of Southern blot hybridizations with the T-DNA of the mannopine type A. rhizogenes 8196. The presence in the plant genome of the pRi-1855 sequences thus identified is demonstrated in carrot roots derived from infection with strain 1855. The T-DNA of pRi-1855 has been mapped by means of cloned Eco RI partial digests. Although strongly homologous with each other, the cores of the T-regions of the mannopine and agropine Ri plasmids are not colinear since the latter contains a central segment of DNA which is absent from the T-region of pRi-8196. Unexpected homologies between the T-region of pRi-1855 and normal carrot DNA have been observed and are discussed here.

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Abstract: Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA3). GA3 greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably α-amylase, and decreasing others. α-Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA3. There was the expected increase in α-amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.

Heat shock proteins and effects of heat shock in plants.

Abstract: Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.

Mitochondrial gene expression and cytoplasmic male sterility in sorghum.

Abstract: Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.

Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

Abstract: The cyanogenic β-glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.

Conservation of ribosomal RNA gene arrangement in the mitochondrial DNA of angiosperms.

Abstract: In six different angiosperms, mitochondrial genes for 18S and 5S rRNAs were found to be closely linked but distant from mitochondrial 26S rRNA genes.

Studies on the structure of cointegrates between octopine and nopaline Ti-plasmids and their tumour-inducing properties.

Abstract: Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens.

Abstract: The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors.

Abstract: Poly (A)+ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (35S)-cysteine and (3H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.

In vivo synthesis and processing of cereal lectins.

Abstract: The synthesis and processing of cereal lectins was followed in vivo. The initial translation products of lectin genes are higher molecular weight (28 K) precursors, which are post-translationally processed in a single step into authentic lectin polypeptides (23 K). The conversion of precursor into mature product is a rather slow process (the precursor has a half life of 36 min) and is apparently not a prerequisite for biological activity since the precursor exhibits sugar binding activity. Because of the striking resemblances between the processing of cereal lectins and vectorial processing of cytoplasmatically made chloroplast, mitochondrial and glyoxysomal proteins, vectorial processing of cereal lectins might be a means of transporting these proteins through a membrane into an extra-cytoplasmic compartment.

The lysopinedehydrogenase gene used as a marker for the selection of octopine crown gall cells.

Abstract: Plant cells transformed into octopine-synthesizing tumour cells by the bacterium Agrobacterium tumefaciens survive when cultured in the presence of homo-arginine (HA), whereas both normal plant cells and nopaline producing plant tumour cells do not. Survival of octopine crown gall cells is due to the activity of the enzyme lysopinedehydrogenase (LpDH) in these cells, which converts toxic homo-arginine into non-toxic homo-octopine. The selective toxicity of homo-arginine for normal cells can be applied for the enrichment of octopine Ti plasmid transformed plant cells vs normal plant cells in mixed cultures.

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza.

Abstract: With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.

Expression of Rhizobium meliloti genes for nitrogen fixation in Escherichia coli minicells: Mapping of the subunit of the nitrogenase reductase

Abstract: An EcoRI fragment of Rhizobium meliloti M2011 which shows homology to Klebsiella pneumoniae DNA carrying nifH and nifD was cloned in both orientations into the Cm gene of plasmid pACYC184 and expressed in Escherichia coli minicells. Fragment specific polypeptides of Mr 12 500, 21 000, 30 000, and 31 000 could be identified. By transposon mutagenesis it was shown that two of them (Mr 12 500 and 21 000) are fusion products with parts of the chloramphenicol acetyltransferase. The other two polypeptides are specified by one coding region which could be mapped by transposon mutagenesis. There are several reasons (homology to Klebsiella nifH, sequence data and molecular weight of the gene products) to assume that this coding region represents the R. meliloti nifH gene (gene for the subunit of the R. meliloti nitrogenase reductase, RmII).

A simple plant polytene chromosome system, and its use for in situ hybridisation.

Abstract: When the cotyledons of Pisum sativum are cultured in the presence of 2–4D, large populations of cells with polytene chromosomes are stimulated to enter prophase; the use of these chromosome for studies of in situ hybridisation with nucleic acids is described.

Repeat sequence interspersion in coding DNA of peas does not reflect that in total pea DNA.

Abstract: The pattern of sequence organization in the regions of the pea genome near sequences coding for mRNA differs significantly from that in total DNA. Interspersion of repeated and single copy sequences is so extensive that 85% of 1300 nucleotide-long fragments contain highly repetitive sequences (about 5000 copies per haploid genome). However, data presented here demonstrate that sequences which code for mRNA are enriched in the small fraction of fragments which do not contain these highly repetitive sequences. Thus, in contrast to the great majority of other sequences in the genome, most mRNA coding sequences are not located within 1300 nucleotides of highly repetitive elements. Moreover, our data indicate that those repeats (if any) which are closely associated with mRNA coding sequences belong to low copy number families characterized by an unusually low degree of sequence divergence.

The chloroplast DNAs of flax genotrophs.

Abstract: The chloroplast DNA of L. usitatissimum var. “Stormont Cirrus” has been mapped with respect to the recognition sites for the enzymes SalP1, Sst1 and SalG1. It is a circular molecule of about 160 kilobasepairs, with an inverted repeat containing the rDNA. Comparisons between chloroplast DNA of uninduced and induced flax genotrophs show there to be no major structural differences.

Open reading frame in the minus strand of two plus type RNA viruses.

Abstract: Inspection of the nucleotide sequences of the RNAs complementary to the coat protein mRNAs from two plant viruses with a tripartite genome: alfalfa mosaic virus and brome mosaic virus, showed the presence of open reading frames for 138 and 118 amino acids, respectively. A third virus (cowpea chlorotic mottle virus) from the same family (1) does not show this phenomenon. This suggests that if a protein is coded for by the open reading frames it may be not essential for virus multiplication. Alternatively the open reading frames have no coding function but result from structural requirements of the RNAs.