Expression of functional oat phytochrome A in transgenic rice.

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.     

Identification of promoter elements involved in the cytosolic Ca(2+)-mediated photoregulation of maize cab-m1 expression.

Changes in cytoplasmic Ca2+ levels are involved in the regulation of several plant genes. However, to our knowledge, no regions of genes or specific cis elements have been shown to be involved in the regulation of plant gene expression by cytosolic Ca2+ signaling. The maize (Zea mays) gene cab-m1, which encodes a light-harvesting chlorophyll a/b-binding apoprotein, is positively photoregulated in mesophyll cells (MC) but not in bundle-sheath cells (BSC). This gene is highly preferentially expressed in maize MC versus BSC. In situ transient expression assays have revealed that exposure of tissues to ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, blocks the photostimulation of cab-m1 full promoter (-1026 to + 14) activity in MC of leaf segments of dark-grown maize seedlings. EGTA has no effect on expression in BSC. These results suggest that light-induced elevation of the cytosolic Ca2+ concentration in MC is required for the enhancement of cab-m1 expression in MC. Deletion of the sequence from -1026 to -360 completely abolished Ca2+ responsiveness of cab-m1 expression in MC. On the other hand, a 54-bp fragment in the 5' flanking region (-953 to -899 relative to the translation start site) conferred Ca2+ responsiveness on a -359 core promoter: reporter gene, suggesting that Ca2+ signaling is mediated via specific sequences in this short fragment. Furthermore, possible involvement of Ca(2+)-calmodulin in the signal transduction chain for regulating cab-m1 expression was suggested by the results of inhibitor experiments.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005–1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll ab ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R FR. Interpretation of these data is difficult. The depression of the chlorophyll ab ratio by low R FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.Abbreviations FR far-red light - PAR photosynthetically active radiation - Pr, Pfr red- and FR-absorbing forms of phytochrome - Ptot total phytochrome - PhyA (PhyA) gene (encoded protein) for phytochrome - R red light - WL white light - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.C.M.; the production of the transgenic seed was funded by the U.S. Department of Energy (DE-F602-88ER13968) to R.D.V., and by E.I. du Pont de Nemours; Dr. G.C. Whitelam is thanked for the provision of monoclonal antibodies for the immunoblot analyses.     

Analysis of cis-acting elements present in the CD20/B1 antigen promoter.

A genomic clone spanning a large portion of the 5' untranscribed region of the CD20 gene was isolated. Deletion analysis of subcloned fragments identified several regulatory elements. A major positive cis-acting element was localized between base pairs -290/-186. A second positive regulatory element was localized between -454/-280 and negative regulatory elements were present in the region between bp -828/-454. The sequence -280/-186 conferred B cell-specific expression on a heterologous, TATA box containing c-fos promoter. Electrophoretic mobility shift assays with overlapping oligonucleotide probes spanning -280/-186 revealed that a 25-bp probe (-225/-201) bound a nuclear protein present in B cell lines expressing the CD20/B1 antigen but not in Jurkat (T cell), U937 (promonocytic), U251 (glioma), or HeLa cells. To confirm the functional significance of this sequence, a trimer of this region was subcloned into the c-fos promoter containing CAT plasmid. Expression was observed only in BJA-B and HS-Sultan cells but not in CD20/B1- cell lines. This sequence element is also important in phorbol ester-induced CD20 expression in the pre-B cell line BP-697. These results partially characterize several regulatory elements present in the CD20 promoter that are likely important in the B cell-specific expression of the CD20 gene.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

Evidence for bound phytochrome in oat seedlings

Phytochrome is consistently observed in pellets centrifuged from homogenates of etiolated, 5-day-old oat seedlings. The majority of pigment associated with the pellet cannot be removed by buffer washes, nor can appreciable quantities of additional phytochrome be adsorbed onto the sedimented material. Over 70% of phytochrome in the pellet is released by 1% Triton X-100.     

Identification of potential protein regulators bound to the tissue-specific positive and negative cis-acting elements of a green tissue-specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.     

Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA.

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.     

Gelatinase A expression in endothelial cells is regulated by at least two cis-acting promoter elements.

Increased expression of gelatinase A is associated with both angiogenesis and alterations in blood vessel structure. Heart-derived endothelial cells derived from spontaneously hypertensive rats (SHR) were found to express significantly more gelatinase A in culture, both at the protein and mRNA level, than endothelial cells from normotensive Wistar-Kyoto (WKY) rats. Other matrix metalloproteinases, as well as their tissue inhibitors, were not differentially regulated. A 1683 bp gelatinase A promoter fragment linked to a luciferase reporter demonstrated up to 40-fold more activity when transfected into SHR-derived cells versus WKY-derived cells. The promoter region between -1324 and -1272, previously termed RE1, contributed up to a five-fold increase in basal promoter activity in both cells, but contributed only 12% of the promoter activity in SHR-derived cells compared to 85% in WKY-derived cells. In SHR-derived cells, but not in WKY-derived cells, a second region between -1435 and -1375, termed RE2, contributed 60% of the total activity of the 1683 bp promoter fragment. Both electrophoretic mobility shift assays and Southwestern blots demonstrated differences in RE2-specific binding factors in nuclear extracts derived from the two cell types. SHR-derived endothelial cells thus represent a new model system to study the regulation of gelatinase A expression, which itself may contribute to the abnormal vascular structure seen in the SHR.     

cis-acting genomic elements and trans-acting proteins involved in the assembly of RNA viruses

There is now considerable evidence that a specific site (or sites) in the genome of an RNA virus interacts with a viral protein to initiate the assembly of the virus ribonucleoprotein or nucleocapsid. We describe the progress that has been made in defining these elements for a number of different viruses: the togavirus, Sindbis virus; the coronavirus, mouse hepatitis virus; influenza A virus; several retroviruses; and the hepadnavirus, hepatitis B virus. The importance of cis-acting elements in packaging has been established for all of these viruses. For Sindbis virus, specificity in the binding of the RNA element to a region of the viral capsid protein in vitro has also been demonstrated.