Stephen H. Howell

Cytokinins and auxins control the expression of a gene in Nicotiana plumbaginifolia cells by feedback regulation.

Both cytokinin (N6-benzyladenine [BA]) and auxin (2,4-dichlorophenoxyacetic acid [2,4-D]) stimulate the accumulation of an mRNA, represented by the cDNA pLS216, in Nicotiana plumbaginifolia suspension culture cells. The kinetics of RNA accumulation were different for the two hormones; however, the response to both was transient, and the magnitude of the response was dose dependent. Runoff transcription experiments demonstrated that the transient appearance of the RNA could be accounted for by feedback regulation of transcription and not by the induction of an RNA degradation system. The feedback mechanism appeared to desensitize the cells to further exposure of the hormone. In particular, cells became refractory to the subsequent addition of 2,4-D after the initial RNA accumulation response subsided. A very different response was observed when the second hormone was added to cells that had been desensitized to the first hormone. Under such conditions, BA produced a heightened response in cells desensitized to 2,4-D and vice versa. These findings support a model in which cytokinin further enhances the auxin response or prevents its feedback inhibition. The hormone-induced RNA accumulation was blocked by the protein kinase inhibitor staurosporin. On the other hand, the protein phosphatase inhibitor okadaic acid stimulated expression, and, in particular, okadaic acid was able to stimulate RNA accumulation in cells desensitized to auxin. This suggests that hormone activation involves phosphorylation of critical proteins on the hormone signaling pathway, whereas feedback inhibition may involve dephosphorylation of these proteins. The sequence of pLS216 is similar to genes in other plants that are stimulated by multiple agonists such as auxins, elicitors, and heavy metals, and to the gene encoding the stringent starvation protein in Escherichia coli. It is proposed that this gene family in various plants be called multiple stimulus response (msr) genes.

The virulent satellite RNA of turnip crinkle virus has a major domain homologous to the 3' end of the helper virus genome.

RNA C (355 bases), RNA D (194 bases) and RNA F (230 bases) are small, linear satellite RNAs of turnip crinkle virus (TCV) which have been cloned as cDNAs and sequenced in this study. These RNAs produce dramatically different disease symptoms in infected plants. RNA C is a virulent satellite that intensifies virus symptoms when co‐inoculated with its helper virus in turnip plants, while RNA D and RNA F are avirulent. RNA D and RNA F, the avirulent satellites, are closely related to each other except that RNA F has a 36‐base insert near its 3′ end, not found in RNA D. The 189 bases at the 5′ end of RNA C, the virulent satellite, are homologous to the entire sequence of RNA D. However, the 3′ half of RNA C, is composed of 166 bases which are nearly identical to two regions at the 3′ end of the TCV helper virus genome. Hence, the virulent satellite is a composite molecule with one domain at its 5′ end homologous to the other avirulent satellites and another domain at its 3′ end homologous to the helper virus genome. All four TCV RNAs, RNAs C, D and F and the helper virus genome have identical 7 bases at their 3′ ends. The secondary structure of RNA C deduced from the sequence can be folded into two separate domains — the domain of helper virus genome homology and the domain homologous to other TCV satellite RNAs. Comparative sequences of several different RNA C clones reveal that this satellite is a population of molecules with sequence and length heterogeneity.

The appearance of DNA breakage and repair activities in the synchronous meiotic cycle of Lilium

Abstract The appearance of three complementary DNA breakage and repair activities is observed during the zygotene-pachytene stages of meiotic prophase in lily microsporocytes. The appearance of these DNA metabolic activities is correlated in time when homologous chromosomes pair and when a DNA repair replication synthesis is observed. One enzymic activity which appears is an endonuclease that produces repairable single-strand DNA breaks with 5′-hydroxyl termini. The endonuclease is accompanied in its appearance by two DNA repair activities, polynucleotidase kinase and polynucleotide ligase. Polynucleotide kinase can phosphorylate 5′-hydroxyl termini produced by the endonuclease and polynucleotide ligase can join the resulting 5′-phosphate to a juxtapositioned 3′- terminus. It is proposed that these activities compose a breakage and reunion mechanism responsible for genetic recombination activities in meiosis.

Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa).

Cauliflower mosaic virus DNA cloned in the Sal I site of bacterial plasmid pBR322 infects turnip plants. The cloned viral DNA must be excised from the recombinant plasmid to infect, but need not be circularized and ligated in vitro. The cloned viral DNA lacks site-specific single-strand breaks found in DNA obtained directly from the virus. However, these breaks are reintroduced into the viral genome during multiplication of the virus in the plant host.

Structure of the Chlamydomonas reinhardtii cabII-1 gene encoding a chlorophyll-a/b-binding protein

Gene cabII-1 is a light regulated gene that encodes the precursor of a major chlorophyll-a/b-binding protein in Chlamydomonas reinhardtii. It is a member of a small gene family composed of about 3-7 members. Nucleotide sequencing data and S1 mapping reveal that the cabII-1 gene is interrupted by three introns. Except for the transit peptide and the N-terminus, the cabII-1 gene product is similar to cabII proteins in higher plants. The cabII-1 gene in C. reinhardtii appears to be an intermediate between type-I and type-II cabII genes described in higher plants.

The identification, mapping, and characterization of mRNA for P66, a cauliflower mosaic virus-coded protein

Turnip leaves infected with cauliflower mosaic virus accumulate variable amounts of a nonvirion protein of 66,000 MW. Tryptic fingerprint analysis showed that this protein is probably not related to the major virus coat protein (37,000 MW). RNA extracted from infected leaves directs the synthesis of a 66,000 MW protein, P66, in a wheat germ protein-synthesizing system. P66 synthesis in vitro could be arrested by prior hybridization of infected leaf RNA to viral DNA restriction fragments indicating that P66 is encoded by the virus genome and specifically by EcoRI fragment b. RNA coding for P66 sediments at about 18 S, binds, in part, to oligo dT-cellulose, and is sensitive to 7-methyl-GTP inhibition.

Regulation of Light-Harvesting Chlorophyll-Binding Protein (LHCP) mRNA Accumulation during the Cell Cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.

THE INTERGENIC REGION OF MAIZE STREAK VIRUS CONTAINS A GC-RICH ELEMENT THAT ACTIVATES RIGHTWARD TRANSCRIPTION AND BINDS MAIZE NUCLEAR FACTORS

Maize streak virus (MSV) is transcribed bidirectionally from an intergenic region and rightward transcription produces an RNA that encodes the coat protein. The intergenic region contains promoter elements required for rightward transcription including an upstream activating sequence (UAS) which endows the promoter with full activity in a maize transient expression system. The UAS contains two GC-rich repeats (GC boxes) and a long inverted repeat or hairpin with a loop harboring a TAATATTAC sequence common to all geminiviruses. Deletions through the UAS demonstrated the presence of an element, called the rightward promoter element (rpe1), which is responsible for transcriptional activation. Rpe1 includes the two GC-rich boxes, which are similar in sequence to Sp1 binding sites in mammalian cells, but not the conserved hairpin loop. Rpe1 binds maize nuclear factors in vitro and the characteristics of the binding interaction have been determined by 1) binding competition with oligonucleotides, 2) methidiumpropyl-EDTA footprinting and 3) methylation interference assays. Binding of maize nuclear factors to the UAS generates two major bands, slow and fast migrating bands, in gel retardation assays. Footprinting and factor titration data suggest that the fast bands arise by the binding of factors to one GC box while the slow bands are generated by factors binding to both boxes. The data further indicate that the factors bind to the two GC-rich boxes with little cooperativity and bind on opposite faces of the DNA helix.

The intergenic region of maize streak virus contains promoter elements involved in rightward transcription of the viral genome

Maize streak virus (MSV), a geminivirus with a one‐component genome, encodes a major coat protein RNA which accumulates in infected plants. Using a maize protoplast cell transient expression system, we have defined and studied the promoter which drives rightward transcription of the RNA encoding the coat protein. We have identified a 122 bp upstream segment that enhances promoter activity and functions as an upstream activating sequence (UAS). The UAS lies in the starting intergenic region of the viral genome and includes a region which is similar in all geminiviruses. The 122 bp UAS activates the MSV core promoter in an orientation, but not position, independent fashion. The MSV promoter UAS is interchangeable with a similar element in the cauliflower mosaic virus (CaMV) 35S RNA core promoter, that is the MSV UAS will activate the CaMV 35S core promoter and vice versa. However, the MSV promoter UAS specifically binds proteins in maize nuclear extracts which appear to differ from those bound by the functionally equivalent region of the CaMV 35S promoter.

Informational complexity of the nuclear and chloroplast genomes of Chlamydomonas reinhardi.

DNA - DNA reassociation kinetics analyzed by hydroxylapatite chromatography have been used to determine the informational content of the Chlamydomonas reinhardi nuclear and chloroplast genomes. The kinetics indicate that nuclear DNA, with the exception of ribosomal cistrons, renatures as a single component with an informational complexity 25 times that of the Escherichia coli genome. The chloroplast genome has less than 0.3% of the informational complexity of the nuclear genome, but is present in about 50 copies in the vegetative cell. Chloroplast DNA shows about a 10-12% zero-time binding component.