Gretchen Hagen

Auxin-regulated gene expression in intact soybean hypocotyl and excised hypocotyl sections.

A library of complementary DNA (cDNA) clones has been prepared from polyadenylated RNA (poly(A)+RNA) from auxin (2,4-dichlorophenoxyacetic acid)-treated soybean (Glycine max (L.) Merr. cv. Wayne) seedlings. Using differential hybridization, four clones were selected as auxin-responsive, and characterized. The levels of the RNA sequences homologous to the cDNA clones were examined in the hypocotyl of the intact seedling and in excised hypocotyl sections before and after auxin treatment, using RNA blot hybridization analysis. RNA levels are rapidly increased (within 0.25–0.5 h) following auxin treatment and the response in the hypocotyl of the intact seedling is transient, reaching maximum RNA levels 2–4 h after auxin application. Increases in RNA levels were also observed with the auxins indole 3-acetic acid and 2,4,5-trichlorophenoxyacetic acid, but not with the ethylene-producing compound, Ethephon (2-chloroethylphosphonic acid). Hybridization analysis of in-vitro transcription products made in nuclei isolated from untreated and auxin-treated soybean primary leaves and excised hypocotyl sections indicates that, for the two cDNA clones analyzed, the increased RNA levels in auxin-treated organs are at least partially the result of increased transcriptional activity of specific DNA sequences.

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight (greater than 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.

A transcriptionally active, covalently closed minichromosome of cauliflower mosaic virus DNA isolated from infected turnip leaves

Purified nuclei from turnip leaves infected by cauliflower mosaic virus (CaMV) have been shown to contain a fraction of CaMV DNA that consists of covalently closed circular molecules; possesses a nucleosome structure, based on sensitivity to micrococcal nuclease; and contains nuclear RNA polymerase II that selectively transcribes the coding strand of CaMV DNA in vitro. Our results suggest that the transcriptionally active CaMV DNA is in the form of a minichromosome and that this DNA does not contain the site-specific discontinuities characteristic of the virion.

The Control of Gene Expression by Auxin

Auxin has been shown to be involved in a variety of diverse plant growth and developmental responses including cell elongation, cell division and cell differentiation (24). The mechanism(s) by which auxin affects such diverse processes is unknown, but, over the past 30 years, several major hypotheses have been proposed and have directed the efforts of auxin biologists. These hypotheses will be discussed briefly as an introduction to a discussion of a current molecular approach being taken to elucidate the mechanism of auxin action.

Two-dimensional gel analysis of the zein proteins in maize.

Abstract The level of complexity of the polypeptide components in the zein (ethanol soluble) fraction from maize kernels can be shown clearly by an analysis that combines the techniques of isoelectric focusing and SDS gel electrophoresis. This high resolution system gives unique and repeatable gel patterns of the zein fraction from 6 maize lines. The gel patterns of zein from normal lines are very complex with over 10 components resolved. The patterns from lines containing the o2 mutation are less complex, with a reduction in the number of components that can be resolved, especially within the 22 500 mol. wt class.

Cloned genomic segments of Zea mays homologous to zein mRNAs

Abstract A maize genome library was constructed using maize W22 DNA from leaf tissue nuclei and bacteriophage λCh4 as the vector. cDNA clones of zein mRNA were used to identify homologous genomic sequences in the Ch4 maize library. Each of the genomic clones identified has homology to a family of mRNAs in the zein mRNA population. This paper reports on the construction of the library, the isolation of the genomic clones and their partial characterization.

The Cauliflower Mosaic Virus Minichromosome

Plant viruses which contain DNA genomes have recently gained popularity as potential vehicles for introducing foreign DNA into plants (reviewed by Howell, 1982; Hohn et al., 1982). The most intensively studied of these DNA viruses is cauliflower mosaic virus (CaMV) which contains a double-stranded DNA genome of about 8 kilobases. The infective DNA purified from virus particles is circular, but not covalently closed (i.e., generally two to three site-specific discontinuities occur in the genome; Hull and Howell, 1978; Volovitch et al., 1978). One of the discontinuities occurs in the α-strand of CaMV DNA and is generally considered as map position 0/1 on the circular genome. The complementary or β-strand of CaMV DNA usually contains two discontinuities at map positions 0.20 and 0.53. In one case, however, the CM4-184 isolate of CaMV contains a deletion of about 421 base pairs in and around map position 0.20 which eliminates one of the discontinuities in the α-strand. In the virus particle, the DNA is surrounded by a protein capsid shell (reviewed by Hohn et al., 1982), but additional proteins such as histones or other chromosomal proteins are not associated with the encapsidated DNA.

[54] – Tissue-Print Hybridization for Detecting RNA Directly

Publisher Summary Tissue printing is a simple method for detecting macromolecules blotted directly from the surfaces of severed organs onto nylon or nitrocellulose membranes. The blotting procedure produces an image of the cut surface of the tissues on the membrane. Macromolecules such as proteins, complex carbohydrates, and nucleic acids are fixed to the membrane. The retention of nucleic acids on the membrane allows the detection of RNAs by hybridization with either DNA or antisense RNA probes. For tissue printing, a dry nylon membrane is placed over a single layer of dry whatman paper or some other absorbent paper. Organs or organ sections are prepared for printing onto membranes by sectioning through the organ with a single- or double-edged razor blade. The freshly cut surfaces are pressed immediately to the nylon membrane or lightly blotted with kimwipes prior to blotting to the membrane. Tissue printing is performed by using firm pressure with the index finger above the sectioned organ for 30–120 sec. The quality of the tissue prints is evaluated by examining the printed nylon membrane under a UV light source. Under UV light, it is possible to observe whether any organ sections were crushed or distorted during blotting. It is found that the large organs of firm consistency such as coytledons, stems, and petioles are much easier to tissue print than small or less firm organs such as roots, leaves, or floral parts.