Showing papers in "Plant Molecular Biology Reporter in 1991"

Nuclear DNA content of some important plant species

Abstract: Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and the horticultural plants should facilitate molecular studies of these crops.

Estimation of nuclear DNA content of plants by flow cytometry

Abstract: A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO4 buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.

A modified CTAB DNA extraction procedure for Musa and Ipomoea

Abstract: The utilization of current nucleic acid technologies in crop improvement and phylogenetic studies require the development and application of efficient DNA extraction procedures from plants. This paper describes efficient, reliable DNA extraction procedures forIpomoea andMusa. These procedures are combinations and modifications of the techniques described by Murray and Thompson (1980) and Saghai-Maroof et al. (1984) and are applicable, without further modification, to other plant genera.

DNA amplification fingerprinting: A strategy for genome analysis

Abstract: A novel strategy to detect genetic differences among organisms, DNA amplification fingerprinting (DAF), uses a thermostable DNA polymerase directed by usually one short (≥5 bp) oligonucleotide primer of arbitrary sequence to amplify short segments of genomic DNA and generate a range of DNA extension products. These products can be analyzed by polyacrylamide gel electrophoresis and silver staining. DAF is rapid and sensitive and is independent of cloning and prior genetic characterization. Here we describe this new methodology, its application to plant genotyping, and its perspectives in DNA fingerprinting and genome mapping.

A rapid means of sex identification inSilene latifolia by use of flow cytometry

Abstract: Sex identification in dioecious plants using nonflowering material would have broad applications in both basic and applied research. We present a method using flow cytometry for diagnosing the sex of the dioecious speciesSilene latifolia Poiret (Caryophyllaceae) by means of sexual differences in nuclear DNA content and base-pair composition. Males have a significantly larger genome, attributable to the known sex-chromosome heteromorphism. Males and females also differ in the AT/GC composition, attributable to differences in non-recombining portions of the sex chromosomes. The two measures enable assignment of individuals to sex with a combined error rate of 9%. These results forS. latifolia indicate useful directions for future research into sex diagnostics for other dioecious species.

Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Abstract: Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.

A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species

Abstract: Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality, restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems.

CHLPEP—A database of chloroplast transit peptides

Abstract: Transit peptides are N-terminal extensions that serve to route nuclearencoded proteins into chloroplasts. We have collected amino acid sequences for approximately 260 transit peptides, and have constructed a database that presently runs under FileMaker Plus on Macintosh personal computers. In addition, the collection is available as an ASCII-file, either in a tab-delimited format suitable for import into other database programs, or in a format with line-identifiers similar to the widely used Swiss Prot database. We hope that the plant molecular biology community will help us ensure that the collection is kept up to date by submitting new sequences directly to us.

Isolation of a sequence common to A- and B-chromosomes of rye (Secale cereale) by microcloning

Abstract: The techniques of microdissection and microcloning have been applied to the isolation of B-chromosome DNA from rye. We have identified a DNA sequence on the rye B-chromosome which is homologous to an A-chromosome sequence, and which is dispersed and moderately repeated on the A- and B-chromosomes. This demonstrates that the rye B-chromosome is heterogeneous in the nature of its DNA sequence composition, containing sequences which are present on the A-chromosomes in addition to those not present on the A-chromosomes.

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

Abstract: We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 106 clones from as little as 1 μg of total RNA.

A method of extraction of DNA from birch

Abstract: A method of DNA extraction is given which is suitable for use with birch (Betula spp.) material collected from natural populations. Such material is frequently old and insect-damaged, and contains high levels of polyphenols. The method relies on repeated extraction of the material with a high molarity urea phosphate buffer, and yields DNA suitable for RFLP analysis.

A consensus sequence of plant hemoglobins

Abstract: From the study of plant hemoglobin protein and gene sequences, a consensus sequence was constructed and analysed by computer methods; analysis comprised the amino acid composition, hydropathy profile, and match degree with other plant and non-plant hemoglobin sequences. Resulting consensus sequence shows the main features of hemoglobin genes and proteins in plants.

A modified β-glucuronidase gene: Sensitive detection of plant promoter activities in suspension-cultured cells of tobacco and rice

Abstract: A translational initiation sequence, conserved among soybean lipoxygenase genes, was fused with the β-glucuronidase (GUS) gene, placed under 35S cauliflower mosaic virus promoter control, then electroporated into suspension-cultured protoplasts of tobacco and rice. These constructs produced 11- and 7-fold increases in GUS activity, respectively, compared with the original GUS construct. The transcriptional activity of the modified GUS gene fused with the TATA box from a soybean lipoxygenase gene was also detected with great sensitivity. The fusion construct is useful for detectingcis-regulatory element effects on transcription, in particular, silencers.

Amplification of a GC-rich sequence from barley by a two-step polymerase chain reaction in glycerol

Abstract: PCR amplification of GC-rich sequences may fail due to poor denaturation or secondary structures that block elongation. Successful amplification of a 672-bp sequence encoding the barley α-amylase/subtilisin inhibitor (69% GC) was achieved in a simple two-step procedure with the addition of 20% glycerol and a high annealing temperature. This protocol may be useful for the amplification of other GC-rich sequences.

Transient gene expression in electroporated bean cotyledon protoplasts

Abstract: A protocol has been developed for the introduction of foreign DNA into cell protoplasts from immature bean cotyledons. The method yields high amounts of the reporter enzymes β-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) when cognate genes are driven by the promoter and upstream sequences of a bean β-phas gene. Comparisons with expression in stable tobacco transformants indicate that transient assays can be used to investigate enhancer function. This techniques should be applicable to other gene systems as well.

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Abstract: Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts

Oligolabeling DNA probes to high specific activity with sequenase

Abstract: In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes produced by this method had a specific activity of 2.8×109 cpm/μg with 69% of the total radioactivity incorporated into the TCA-precipitable materials. Probes with 5 to 10-fold lower specific activity were obtained using commercially available kits or using currently empolyed methods.

Monitoring hairy-root growth by image analysis

Abstract: Hairy roots, incited byAgrobacterium rhizogenes, form a useful system for analysing the expression and phenotypic effects of foreign genes in plant root tissue. Image analysis offers a non-invasive method of describing their growth in culture. Images of pea (coarse) andBrassica (fine) hairy roots were captured, processed and analysed without difficulty using a commercially available image analysis system. The value of this method in monitoring intermediate changes in growth pattern was illustrated by following the changes in five putatively chlorsulfuron-resistantBrassicc hairy-root lines cultured with and without a selective level of chlorsulfuron. Areas of hairy-root research where this technique will be particularly useful are discussed.

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

Abstract: A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.

On naming plant genes

Abstract: T he ISPMB has received a small start-up grant from the U. S. National Science Foundation to begin development of systems and procedures for collating, storing and disseminating the data generated by the world wide plant genome mapping/sequencing efforts. The principal investigator for this grant is Carl Price of Rutgers University, Editor of the Plant Molecular Biology Reporter. His co-principal investigator for informatics is Gregory Harem, also of Rutgers, who founded the EMBL Library of DNA sequences some years ago. Harem and his colleagues, who are assessing the requirements for a database for plant genomes, have pointed out that an early requisite for their work is a universally recognized system for naming plant genes. Since no such system exists, Price has appointed, in the name of the ISPMB, an international Commission on Plant GeneNomenclature (PMBR 9(2):103, 1991). Further, he has solicited ideas for the construction of such a system from the world at large. Despite the likelihood of appearing silly, foolish, p resumptuous and / or ignorant, I venture to offer an outline of a system for the naming of plant genes. Further, I have found it hard in recent weeks to resist thinking about the nomenclature problem every time I take a shower. Thus, I am obliged to get these thoughts on paper, so I can think about other things. As a preamble to the system itself, the following is a list of caveats and considerations that may be useful in putting the genenaming enterprise in perspective.

ADPG revisited—Nearly 30 years from Buenos Aires and Los Baños to Nagoya

Abstract: A N article by Art Weissbach and Bernie Horecker (1989) on their pioneering work on RuBisCo reminded me of our study oll RuBisCo in leaves of rice (Mendiola and Akazawa, 1964) carried out at the International Rice Research Institute (IRRI) at Los Ba f~os in The Philippines. I believe that our work showed quite convincingly that the reaction product of CO 2 fixation catalyzed by the rice leaf \"fraction 1 protein\" was 3-PGA. [Wildman and Bonner (1947) described \"fraction I protein\" as the major soluble protein in leaves; its enzymatic role as RuBisCo was discovered much later.] Instead of describing our work on RuBisCo, however, I should like to offer the background story of our research on ADPG and starch biosynthesis initiated at IRRI. ADPG was not as fashionable as RuBisCo, but recent advances in our laboratory concerning the transport of ADPG across the plastid envelope and its relation to the gluconeogenic pathway brings me back to the youthful days in the Philippines nearly 30 years ago. On this occasion 1 wish to record my sincere thanks to Robert F. Chandler, Sterling Wortman, and other staff members of IRRI, who warmly supported and encouraged us to conduct fundamental research work using tropical rice (1962 to 1964). I fondly confess that much of the current research in my laboratory has its roots in those bygone days. Soon after my arrival at IRRI, I decided to work on the mechanism of starch biosynthesis in rice seeds. At that time a group in Buenos Aires

Construction of a deletion-scanning library for the 5′-proximal promoter region of the bean β-phaseolin gene

Abstract: A library of internal deletion mutants was constructed in the 5′-proximal promoter region of the gene encoding the bean seed storage protein β-phaseolin. A nick was introduced randomly in the target DNA sequence by depurination and treatment with exonuclease III, and served as the initiating point for deleting adjacent DNA sequences by S1 nuclease. A syntheticPst I linker was ligated to the blunt-ended DNA to serve as a restriction marker for mapping the approximate position of deletion mutants. Subcloning of a kanamycin marker gene into the linker site facilitated selection of plasmid DNA in which internal deletions were introduced in the target DNA sequence. Distribution of internal deletion mutants was mapped by determining the locations of thePst I site in the target sequence. DNA sequence analysis of mutants indicated that the extent of internal deletions ranged from 6 to 100 bp with a mean of 39 bp. In addition, the CAAT and TATA boxes in the promoter region of the β-phaseolin gene were effectively dissected in these mutants.