Elizabeth A. Davis

A Putative CCAAT-Binding Transcription Factor Is a Regulator of Flowering Timing in Arabidopsis

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.

Partial purification of plant transcription factors. II. An in vitro transcription system is inefficient

Crude wheat germ nuclear extracts contain many inhibitors of transcription which need to be removed before an active system can be developed. Using ion exchange column chromatography to resolve RNA polymerase II transcription components we can identify at least four fractions required for transcription by their ability to interact with, or substitute for, particular HeLa fractions. Inhibitors can be removed by a second or third chromatographic process applied to each fraction. Two plant fractions can each effectively replace the corresponding fraction in a HeLa transcription system, and the wheat fractions can work together and replace two HeLa fractions. These plant factors chromatograph identically to HeLa factors on ion exchange columns. The third fraction does not fully substitute for the corresponding HeLa fraction, but can complement this HeLa fraction when both are added at half-optimal levels. An in vitro plant system consisting of four plant chromatographic fractions will selectively transcribe a gene, but only at very low efficiency. The apparent block to greater efficiency is in elongation of the RNA past the 20–30n size.

Coactivators and TAFs of transcription activation in wheat

Transcription regulation often activates quiescent genes in a tissue-specific or developmental manner. Activator proteins bind to a DNA sequence upstream of the promoter, interact with the general transcription proteins via bridging proteins, and elevate transcription levels. One group of bridging proteins, the coactivators, have been characterized in animals as polypeptides tightly associated with the general transcription factor TATA-binding protein (TBP). They are referred to as TAFs (TBP-associated factors), and together with TBP comprise general transcription factor IID. We provide biochemical evidence that wheat IID contains coactivators. An activator protein with an acidic activation domain facilitates the binding of IID to the template, and potentiates activated in vitro transcription with wheat IID, but not with wheat TBP. Using antibodies to wheat TBP, we demonstrate that wheat IID also contains TAFs. This is the first demonstration that a plant contains coactivators and TAFs.

Partial purification of plant transcription factors. I. Initiation.

Crude plant cell protein extracts prepared from wheat germ are inactive for in vitro transcription by RNA polymerase II. These extracts do, however, have correct initiation of transcription by RNA polymerase II. Initiation is monitored by measuring the formation of transcription complexes in vitro. A nuclear extract produces more initiation events than a whole cell extract or a cytosol extract. Some factors necessary for initiation can be separated from other proteins, including inhibitors, by ion exchange column chromatography. One specific fraction is sufficient for the formation of transcription complexes and several other fractions may be stimulatory or accessory factors.

Immunohistochemical detection of polychlorinated biphenyls in field collected damselfish (Abudefduf sordidus; Pomacentridae) embryos and larvae

Antibodies against polychlorinated biphenyls (PCBs) were used to determine if immunohistochemical methods could detect PCBs in embryos and larvae of a territorial coral reef fish (Abudefduf sordidus; Pomacentridae) collected from Johnston Atoll, Central Pacific Ocean. Sites with differing levels of contamination were sampled, one with relatively high sediment PCB concentrations of up to 389.0 ng/g and another with low PCB concentrations of only 0.5 ng/g. Immunostaining suggested that PCB concentrations were higher in fish larvae from the PCB contaminated site and that PCB concentrations within abnormal embryos were higher than normal embryos from the same nest. This technique will be useful for detecting exposed populations in the field and assessing correlations with adverse effects, particularly in potential indicator organisms such as Abudefduf sordidus.

RNA polymerase II transcription complexes are destabilized by ATP or GTP

In vitro transcription by RNA polymerase II requires hydrolysis of the beta-gamma bond of ATP after the transcription complex forms, prior to RNA synthesis. It was observed that the presence of ATP during transcription complex formation inhibits subsequent transcription when the remaining 3 rNTPs are added. We now report that ATP or GTP inhibits transcription if either is present during transcription complex formation to added to preformed complexes. This inhibition is not due to purine rNTP degradation and occurs if as little as 2 mM ATP or 50 mM GTP is added to forming or preformed complexes. Deoxy derivatives of ATP inhibit similarly. AMP-PNP, a beta-gamma imido derivative, neither satisfies the energy requirement nor inhibits transcription if added to incubations of forming or of preformed transcription complexes.

Transcription factor IIA of wheat and human interacts similarly with the adenovirus-2 major late promoter

Transcription factor IIA (TFIIA) is a necessary component of many RNA polymerase II transcription complexes. Assembly of the transcription complex begins when TFIIA interacts with the promoter. We have previously purified wheat germ TFIIA to homogeneity and demonstrated that it substitutes for human TFIIA in a human in vitro transcription system which utilizes the adenovirus-2 major late promoter (Ad-2 MLP). We now show, by gel retardation assays, that wheat TFIIA interacts with the Ad-2 MLP. Extensively purified human (HeLa) TFIIA interacts with the Ad-2 MLP similarly. Both wheat and human TFIIA interact with a DNA fragment comprising the minimal promoter region (-51/+32) but not with upstream or downstream regions. With both TFIIAs multiple complexes form; the fastest wheat TFIIA/DNA complex appears to be larger than the corresponding human TFIIA/DNA complex. Limited point mutation analysis of the Ad-2 MLP demonstrates that changes at -30 (TATAA region), +1, and -1 diminish TFIIA binding, but a change at -40 does not. DNA footprint analysis of this region is not definitive, but does indicate that following TFIIA binding there are changes in the pattern of hypersensitive sites.

Alterations in Photosynthetic Pigment Synthesis in Tissue Cultured Tobacco Callus

Tobacco stem pith in tissue culture forms green callus, followed by shoot regeneration. Inclusion of excess manganese (10 mM) in the medium leads to inhibition of growth and of photosynthetic pigment biosynthesis. Growth is inhibited about 40% throughout the 7 week period of study. Pigment synthesis is initially delayed in manganese treated cultures, then the extent of inhibition decreases. The amounts of lutein and β-carotene relative to chlorophyll a. are reduced during the first few weeks of manganese treatment7 while the relative amount of chlorophyll b remains the same. During later stages of manganese treatment, both lutein and chlorophyll b increase relative to chlorophyll a.