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Papers by Anthony Cashmore

Research paper thumbnail of Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development

The Plant Journal, 1996

Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates ... more Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV-A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light-dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV-A, and green light in a fluence rate-dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light-induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light-dependent regulation of gene expression in addition to its affect on plant growth.

Research paper thumbnail of The CRY1 Blue Light Photoreceptor of Arabidopsis Interacts with Phytochrome A In Vitro

Molecular Cell, 1998

The cryptochrome photoreceptors are evolutionarily ancient, with homologs identified in numerous ... more The cryptochrome photoreceptors are evolutionarily ancient, with homologs identified in numerous plant species including the green alga

Research paper thumbnail of The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

The Plant Journal, 1997

et al., 1995a; Malhotra et al.,1995), and overexpression of USA CRY1 protein in transgenic plants... more et al., 1995a; Malhotra et al.,1995), and overexpression of USA CRY1 protein in transgenic plants conferred a blue-light hypersensitive phenotype (Lin et al.,1995b), consistent with its role as photoreceptor. Blue-light-dependent phenotypes Summary shown to be under the control of CRY1 include inhibition Blue-light responses in higher plants are mediated by of hypocotyl elongation and anthocyanin production in specific photoreceptors, which are thought to be flavoseedlings (Ahmad et al., 1995; Jackson and Jenkins, 1995; proteins; one such flavin-type blue-light receptor, CRY1 Koornneef et al., 1980). In spite of its striking homology to (for cryptochrome), which mediates inhibition of hypocotyl the DNA photolyases, CRY1 shows no demonstrable DNA elongation and anthocyanin biosynthesis, has recently binding or photoreactivating activity (Lin et al., 1995a; been characterized. Prompted by classical photobiological Malhotra et al., 1995). The structure of CRY1 suggests a studies suggesting possible co-action of the red/far-red mechanism of action involving electron transfer; the reacabsorbing photoreceptor phytochrome with blue-light tion partners and downstream transduction apparatus photoreceptors in certain plant species, the role of phytoremain to be identified. chrome in CRY1 action in Arabidopsis was investigated. A recurring theme in plant blue-light research has been The activity of the CRY1 photoreceptor can be substantially an involvement of the red/far-red-absorbing photoreceptor altered by manipulating the levels of active phytochrome phytochrome in physiological responses to blue-light treat-(Pfr) with red or far-red light pulses subsequent to bluements. Experiments in a number of monocot and dicot light treatments. Furthermore, analysis of severely phytoplant species have shown that blue-light responses such chrome-deficient mutants showed that CRY1-mediated as inhibition of hypocotyl elongation or anthocyanin accublue-light responses were considerably reduced, even mulation can be partially reversed if the blue-light pulses though Western blots confirmed that levels of CRY1 photoare followed by, or given in the presence of, saturating receptor are unaffected in these phytochrome-deficient pulses of far-red light (Casal, 1994; Gaba et al., 1984; mutant backgrounds. It was concluded that CRY1-medi-Mancinelli et al., 1991; Mohr, 1994). Such far-red reversiated inhibition of hypocotyl elongation and anthocyanin bility had been taken as evidence that phytochrome, or the production requires active phytochrome for full expresphytochrome signal transduction pathway, was somehow sion, and that this requirement can be supplied by low implicated in blue-light responses. However, interpretation levels of either phyA or phyB. of these studies has been complicated by the fact that the phytochrome photoreceptor itself directly absorbs blue light. It is therefore difficult to unequivocally distinguish

Research paper thumbnail of Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light

Proceedings of the National Academy of Sciences, 1995

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, enc... more The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery fo...

Research paper thumbnail of Cryptochromes

Cell, 2003

can alter the phase, the time of peak activity, of these rhythms. The molecular basis of this rhy... more can alter the phase, the time of peak activity, of these rhythms. The molecular basis of this rhythmic activity commonly involves a transcriptional feedback loop; the positive and negative regulatory components of these loops have been the subject of intense study (Harmer

Research paper thumbnail of Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Journal of Biological Chemistry, 1983

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic r... more Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) h c. NutL A& Sci U. S. A. 78,7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NHz-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAl396) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.

Research paper thumbnail of Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins

The EMBO Journal, 1992

Communicated by M.van Montagu The promoters of a variety of plant genes are characterized by the ... more Communicated by M.van Montagu The promoters of a variety of plant genes are characterized by the presence of a G-box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G-box sequence has been demonstrated to be essential for expression. The G-box of the Arabidopsis rbcS-IA gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N-terminal proline-rich domain. Homodimers of the three proteins specifically recognize the G-box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G-box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G-box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G-box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.

Research paper thumbnail of The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor

The EMBO Journal, 1990

Communicated by M.van Montagu G box and I box sequences of the Arabidopsis thaliana ribulose-bisp... more Communicated by M.van Montagu G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-IA promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a-390 to-60 rbcS-IA promoter fragment containing the G box and two I boxes activates transcription from a tnmcated iso-l-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-IA G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYCl promoter. Single-copy G box sequences from the Arabidopsis rbcS-JA, Anabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts.

Research paper thumbnail of Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter

The EMBO Journal, 1990

Communicated by M.van Montagu A deletion analysis of the Arabidopsis thaliana rbcS-IA promoter de... more Communicated by M.van Montagu A deletion analysis of the Arabidopsis thaliana rbcS-IA promoter defined a 196 bp region (-320 to-125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Sitespecific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-IA promoter substantially reduced the expression ofAdh and 3-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-IA promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-IA promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter-GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to Gbox-mutated rbcS-IA sequences.

Research paper thumbnail of Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II

Plant Physiology, 1994

Broccoli (Brassica oleracea var italica) was purchased from local wholesale distributors. [y-32P]... more Broccoli (Brassica oleracea var italica) was purchased from local wholesale distributors. [y-32P]ATP (specific activity 3000 Ci/mmol) was obtained from ICN Radiochemicals (Costa Abbreviations: CKI and CKII, casein kinase I and 11; DEAM, diethylaminomethyl; HMG, high-mobility group; MALDI/MS, matrix-assisted laser desorption ionization mass spectrometry. 91 1

Research paper thumbnail of Binding of a pea nuclear protein to promoters of certain photoregulated genes is modulated by phosphorylation

Trends in Genetics, 1990

of at least one of these proteins appears to be modulated by this modification [Montminy and Bile... more of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)l. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of A A T A r m T A l T was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and toses all DNAbinding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.

Research paper thumbnail of Cryptochrome 1 controls tomato development in response to blue light

The Plant Journal, 1999

Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong re... more Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong resemblance to photolyases, a rare class of flavoproteins that absorb light in the blue (B) and UV-A regions of the spectrum and utilise it for photorepair of UV-damaged DNA. In Arabidopsis, both CRY1 and CRY2 are implicated in numerous blue light-dependent responses, including inhibition of hypocotyl elongation, leaf and cotyledon expansion, pigment biosynthesis, stem growth and internode elongation, control of flowering time and phototropism. No information about the in vivo function of CRY genes is available in other plant species. The tomato CRY1 gene (TCRY1) encodes a protein of 679 amino acids, which shows 78% identity and 88% similarity to Arabidopsis CRY1. In order to verify the in vivo function of TCRY1, we constructed antisense tomato plants using the C-terminal portion of the gene. Partial repression of both mRNA and protein levels was observed in one of the transformants. The progeny from this transformant showed an elongated hypocotyl under blue but not under red light. This character co-segregated with the transgene and was dependent on transgene dosage. An additional, partially elongated phenotype was observed in adult plants grown in the greenhouse under dim light and short days with no artificial illumination. This phenotype was suppressed by artificial illumination of both short and long photoperiods. The synthesis of anthocyanins under blue light was reduced in antisense seedlings. In contrast, carotenoid

Research paper thumbnail of TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG

Research paper thumbnail of Chimeric Proteins between cry1 and cry2 Arabidopsis Blue Light Photoreceptors Indicate Overlapping Functions and Varying Protein Stability

The Plant Cell, 1998

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently an... more A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.

Research paper thumbnail of An Enzyme Similar to Animal Type II Photolyases Mediates Photoreactivation in Arabidopsis

The Plant Cell, 1997

The important issue of photoreactivation DNA repair in plants has become even more interesting in... more The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHRl (for @otoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHRl gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHRl is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHRl protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHRl represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.

Research paper thumbnail of Level of Expression of the Tomato rbcS-3A Gene is Modulated by a Far Upstream Promoter Element in a Developmentally Regulated Manner

THE PLANT CELL ONLINE, 1989

By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter seque... more By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter sequence from the tomato rbcS-3A gene confers light-inducible and organ-specific expression upon fusion to the bacterial chloramphenicol acetyltransferase gene. A biphasic expression profile was obtained by 5" deletion analysis of this promoter, indicating the presence of both positive and negative regulatory elements. A severe reduction in the level of expression was observed when the 5'-terminal 90 base pairs were deleted from the 1.10-kilobase promoter. DNA sequence elements responsible for light inducibility and organ specificity of the gene reside within the-374 base pairs of the proximal part of the promoter and the sequences spanning from-374 to-205 are essential for promoter function. The DNA sequences upstream from-374 modulate the level of expression in leaf tissue; this modulation is under developmental control.

Research paper thumbnail of DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli

The Plant Cell, 1992

To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBFl), we have... more To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBFl), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBFl was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylating activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low K, value for GBFl (220 nM compared to-10 pM for casein) was in the range observed for identified physiological substrates of casein kinase 11. Phosphorylation of GBFl resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.

Research paper thumbnail of Reconstitution of Arabidopsis casein kinase II from recombinant subunits and phosphorylation of transcription factor GBF1

The Plant Cell, 1995

Ente per le Nuove tecnologie. I'Energia e IXmbiente (ENEA), CR Casaccia. PO. Box 2400, Rome O0100... more Ente per le Nuove tecnologie. I'Energia e IXmbiente (ENEA), CR Casaccia. PO. Box 2400, Rome O0100 AD, ltaly In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII)? plant CKll is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit a (CKA1) and the regulatory subunit p (CKB1) of CKll were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKBl on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKAl and CKBl proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKAl activity alone, showing that CKBl has biochemical properties similar to those of the 13 subunit from animals. CKAl and CKBl spontaneously assembled into a tetrameric complex, CKA12CKB12, which had properties very similar to those of the oligomeric CKll form isolated from broccoli. However, the properties of the catalytic subunit CKAl alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBFl with the reconstituted CKA12CKB12 enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKll from broccoli.

Research paper thumbnail of Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex

Journal of Cell Biology, 1981

We have used an in vitro reconstitution system, consisting of cell-free translation products and ... more We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursor...

Research paper thumbnail of Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

Proceedings of the National Academy of Sciences, 1984

A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of... more A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80 . The first methionine codon 3′ from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A “TATA” sequence occurs 31 nucleotides 5′ from the cap site. A second TATA sequence is found 7 nucleotides on the 5′ side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within t...

Research paper thumbnail of Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development

The Plant Journal, 1996

Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates ... more Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV-A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light-dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV-A, and green light in a fluence rate-dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light-induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light-dependent regulation of gene expression in addition to its affect on plant growth.

Research paper thumbnail of The CRY1 Blue Light Photoreceptor of Arabidopsis Interacts with Phytochrome A In Vitro

Molecular Cell, 1998

The cryptochrome photoreceptors are evolutionarily ancient, with homologs identified in numerous ... more The cryptochrome photoreceptors are evolutionarily ancient, with homologs identified in numerous plant species including the green alga

Research paper thumbnail of The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

The Plant Journal, 1997

et al., 1995a; Malhotra et al.,1995), and overexpression of USA CRY1 protein in transgenic plants... more et al., 1995a; Malhotra et al.,1995), and overexpression of USA CRY1 protein in transgenic plants conferred a blue-light hypersensitive phenotype (Lin et al.,1995b), consistent with its role as photoreceptor. Blue-light-dependent phenotypes Summary shown to be under the control of CRY1 include inhibition Blue-light responses in higher plants are mediated by of hypocotyl elongation and anthocyanin production in specific photoreceptors, which are thought to be flavoseedlings (Ahmad et al., 1995; Jackson and Jenkins, 1995; proteins; one such flavin-type blue-light receptor, CRY1 Koornneef et al., 1980). In spite of its striking homology to (for cryptochrome), which mediates inhibition of hypocotyl the DNA photolyases, CRY1 shows no demonstrable DNA elongation and anthocyanin biosynthesis, has recently binding or photoreactivating activity (Lin et al., 1995a; been characterized. Prompted by classical photobiological Malhotra et al., 1995). The structure of CRY1 suggests a studies suggesting possible co-action of the red/far-red mechanism of action involving electron transfer; the reacabsorbing photoreceptor phytochrome with blue-light tion partners and downstream transduction apparatus photoreceptors in certain plant species, the role of phytoremain to be identified. chrome in CRY1 action in Arabidopsis was investigated. A recurring theme in plant blue-light research has been The activity of the CRY1 photoreceptor can be substantially an involvement of the red/far-red-absorbing photoreceptor altered by manipulating the levels of active phytochrome phytochrome in physiological responses to blue-light treat-(Pfr) with red or far-red light pulses subsequent to bluements. Experiments in a number of monocot and dicot light treatments. Furthermore, analysis of severely phytoplant species have shown that blue-light responses such chrome-deficient mutants showed that CRY1-mediated as inhibition of hypocotyl elongation or anthocyanin accublue-light responses were considerably reduced, even mulation can be partially reversed if the blue-light pulses though Western blots confirmed that levels of CRY1 photoare followed by, or given in the presence of, saturating receptor are unaffected in these phytochrome-deficient pulses of far-red light (Casal, 1994; Gaba et al., 1984; mutant backgrounds. It was concluded that CRY1-medi-Mancinelli et al., 1991; Mohr, 1994). Such far-red reversiated inhibition of hypocotyl elongation and anthocyanin bility had been taken as evidence that phytochrome, or the production requires active phytochrome for full expresphytochrome signal transduction pathway, was somehow sion, and that this requirement can be supplied by low implicated in blue-light responses. However, interpretation levels of either phyA or phyB. of these studies has been complicated by the fact that the phytochrome photoreceptor itself directly absorbs blue light. It is therefore difficult to unequivocally distinguish

Research paper thumbnail of Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light

Proceedings of the National Academy of Sciences, 1995

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, enc... more The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery fo...

Research paper thumbnail of Cryptochromes

Cell, 2003

can alter the phase, the time of peak activity, of these rhythms. The molecular basis of this rhy... more can alter the phase, the time of peak activity, of these rhythms. The molecular basis of this rhythmic activity commonly involves a transcriptional feedback loop; the positive and negative regulatory components of these loops have been the subject of intense study (Harmer

Research paper thumbnail of Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Journal of Biological Chemistry, 1983

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic r... more Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) h c. NutL A& Sci U. S. A. 78,7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NHz-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAl396) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.

Research paper thumbnail of Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins

The EMBO Journal, 1992

Communicated by M.van Montagu The promoters of a variety of plant genes are characterized by the ... more Communicated by M.van Montagu The promoters of a variety of plant genes are characterized by the presence of a G-box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G-box sequence has been demonstrated to be essential for expression. The G-box of the Arabidopsis rbcS-IA gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N-terminal proline-rich domain. Homodimers of the three proteins specifically recognize the G-box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G-box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G-box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G-box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.

Research paper thumbnail of The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor

The EMBO Journal, 1990

Communicated by M.van Montagu G box and I box sequences of the Arabidopsis thaliana ribulose-bisp... more Communicated by M.van Montagu G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-IA promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a-390 to-60 rbcS-IA promoter fragment containing the G box and two I boxes activates transcription from a tnmcated iso-l-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-IA G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYCl promoter. Single-copy G box sequences from the Arabidopsis rbcS-JA, Anabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts.

Research paper thumbnail of Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter

The EMBO Journal, 1990

Communicated by M.van Montagu A deletion analysis of the Arabidopsis thaliana rbcS-IA promoter de... more Communicated by M.van Montagu A deletion analysis of the Arabidopsis thaliana rbcS-IA promoter defined a 196 bp region (-320 to-125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Sitespecific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-IA promoter substantially reduced the expression ofAdh and 3-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-IA promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-IA promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter-GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to Gbox-mutated rbcS-IA sequences.

Research paper thumbnail of Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II

Plant Physiology, 1994

Broccoli (Brassica oleracea var italica) was purchased from local wholesale distributors. [y-32P]... more Broccoli (Brassica oleracea var italica) was purchased from local wholesale distributors. [y-32P]ATP (specific activity 3000 Ci/mmol) was obtained from ICN Radiochemicals (Costa Abbreviations: CKI and CKII, casein kinase I and 11; DEAM, diethylaminomethyl; HMG, high-mobility group; MALDI/MS, matrix-assisted laser desorption ionization mass spectrometry. 91 1

Research paper thumbnail of Binding of a pea nuclear protein to promoters of certain photoregulated genes is modulated by phosphorylation

Trends in Genetics, 1990

of at least one of these proteins appears to be modulated by this modification [Montminy and Bile... more of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)l. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of A A T A r m T A l T was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and toses all DNAbinding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.

Research paper thumbnail of Cryptochrome 1 controls tomato development in response to blue light

The Plant Journal, 1999

Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong re... more Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong resemblance to photolyases, a rare class of flavoproteins that absorb light in the blue (B) and UV-A regions of the spectrum and utilise it for photorepair of UV-damaged DNA. In Arabidopsis, both CRY1 and CRY2 are implicated in numerous blue light-dependent responses, including inhibition of hypocotyl elongation, leaf and cotyledon expansion, pigment biosynthesis, stem growth and internode elongation, control of flowering time and phototropism. No information about the in vivo function of CRY genes is available in other plant species. The tomato CRY1 gene (TCRY1) encodes a protein of 679 amino acids, which shows 78% identity and 88% similarity to Arabidopsis CRY1. In order to verify the in vivo function of TCRY1, we constructed antisense tomato plants using the C-terminal portion of the gene. Partial repression of both mRNA and protein levels was observed in one of the transformants. The progeny from this transformant showed an elongated hypocotyl under blue but not under red light. This character co-segregated with the transgene and was dependent on transgene dosage. An additional, partially elongated phenotype was observed in adult plants grown in the greenhouse under dim light and short days with no artificial illumination. This phenotype was suppressed by artificial illumination of both short and long photoperiods. The synthesis of anthocyanins under blue light was reduced in antisense seedlings. In contrast, carotenoid

Research paper thumbnail of TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG

Research paper thumbnail of Chimeric Proteins between cry1 and cry2 Arabidopsis Blue Light Photoreceptors Indicate Overlapping Functions and Varying Protein Stability

The Plant Cell, 1998

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently an... more A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.

Research paper thumbnail of An Enzyme Similar to Animal Type II Photolyases Mediates Photoreactivation in Arabidopsis

The Plant Cell, 1997

The important issue of photoreactivation DNA repair in plants has become even more interesting in... more The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHRl (for @otoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHRl gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHRl is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHRl protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHRl represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.

Research paper thumbnail of Level of Expression of the Tomato rbcS-3A Gene is Modulated by a Far Upstream Promoter Element in a Developmentally Regulated Manner

THE PLANT CELL ONLINE, 1989

By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter seque... more By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter sequence from the tomato rbcS-3A gene confers light-inducible and organ-specific expression upon fusion to the bacterial chloramphenicol acetyltransferase gene. A biphasic expression profile was obtained by 5" deletion analysis of this promoter, indicating the presence of both positive and negative regulatory elements. A severe reduction in the level of expression was observed when the 5'-terminal 90 base pairs were deleted from the 1.10-kilobase promoter. DNA sequence elements responsible for light inducibility and organ specificity of the gene reside within the-374 base pairs of the proximal part of the promoter and the sequences spanning from-374 to-205 are essential for promoter function. The DNA sequences upstream from-374 modulate the level of expression in leaf tissue; this modulation is under developmental control.

Research paper thumbnail of DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli

The Plant Cell, 1992

To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBFl), we have... more To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBFl), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBFl was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylating activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low K, value for GBFl (220 nM compared to-10 pM for casein) was in the range observed for identified physiological substrates of casein kinase 11. Phosphorylation of GBFl resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.

Research paper thumbnail of Reconstitution of Arabidopsis casein kinase II from recombinant subunits and phosphorylation of transcription factor GBF1

The Plant Cell, 1995

Ente per le Nuove tecnologie. I'Energia e IXmbiente (ENEA), CR Casaccia. PO. Box 2400, Rome O0100... more Ente per le Nuove tecnologie. I'Energia e IXmbiente (ENEA), CR Casaccia. PO. Box 2400, Rome O0100 AD, ltaly In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII)? plant CKll is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit a (CKA1) and the regulatory subunit p (CKB1) of CKll were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKBl on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKAl and CKBl proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKAl activity alone, showing that CKBl has biochemical properties similar to those of the 13 subunit from animals. CKAl and CKBl spontaneously assembled into a tetrameric complex, CKA12CKB12, which had properties very similar to those of the oligomeric CKll form isolated from broccoli. However, the properties of the catalytic subunit CKAl alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBFl with the reconstituted CKA12CKB12 enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKll from broccoli.

Research paper thumbnail of Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex

Journal of Cell Biology, 1981

We have used an in vitro reconstitution system, consisting of cell-free translation products and ... more We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursor...

Research paper thumbnail of Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

Proceedings of the National Academy of Sciences, 1984

A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of... more A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80 . The first methionine codon 3′ from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A “TATA” sequence occurs 31 nucleotides 5′ from the cap site. A second TATA sequence is found 7 nucleotides on the 5′ side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within t...