A protein factor, identified in nuclear ex-

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab‐E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens‐mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis‐acting elements that influence photoregulated expression of the Cab‐E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence‐ACCGGCCCACTT‐. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from −396 to −186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase (‘nos’) promoter. Within this region there is a 132‐bp element, extending from −368 to −234 bp, which on deletion from the Cab‐E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab‐E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab‐E TATA box are not replaceable by corresponding sequences from a ‘nos’ promoter. This contrasts with the apparent equivalence of these Cab‐E and ‘nos’ TATA box‐proximal sequences in truncated promoters conferring low levels of photoregulated expression.

The photosynthetic apparatus of plant chloroplasts contains two photosystems, termed Photosystem I (PSI) and Photosystem II (PSII). Both PSI and PSII contain several types of chlorophyll a/b-binding (CAB) polypeptides, at least some of which are structurally related. It has been previously shown that multiple genes encoding one type of PSII CAB polypeptides exist in the genome of many higher plants. In tomato, there are at least eight such genes, distributed in three independent loci. Genes encoding a second type of CAB polypeptides have been isolated from several plant species, but the precise location of the gene products has not been determined. Here we show that tomato has two unlinked genes encoding this second type and that this type of CAB polypeptide is also localized in PSII.

We report here the isolation and nucleotide sequence of a complete cDNA clone encoding a photosystem I (PS I) polypeptide that is recognized by a monoclonal antibody made against photosystem II (PS II) chlorophyll a/b-binding (CAB) proteins. The deduced sequence of this PS I protein shows 30% overall identity to PS II CAB sequences, and two long segments within this protein show 50% and 65% identity to the corresponding segments in the PS I CAB polypeptides. Even though the sequence of this PS I CAB protein is substantially divergent from PS II CAB sequences, their hydropathy plots are very similar and suggest they all traverse the thylakoid membrane three times. A segment of the PS I CAB polypeptide shows similarity to the functionally analogous (3 subunits of the antenna proteins of purple bacteria. In contrast, no homology was observed between these bacterial proteins and PS II CAB polypeptides. MATERIALS AND METHODSFractionation of PS I and PS 11. Tomato (Lycopersicon esculentum) PS I and PS II were fractionated according to Mullet et al. (14) with the exception that the Triton X-100 concentration was changed to 0,5% (wt/vol) for the solubilization of the thylakoid membrane. Under these conditions, the chlorophyll concentration was 0.8 mg/ml.Polyacrylamide Gel Electrophoresis. Protein samples were subjected to NaDodSO4/polyacrylamide gel electrophoresis in 13% gels under reducing conditions (13,15 Immunoblots. Protein was transferred to nitrocellulose paper at pH 8.3 by applying a voltage gradient of 30 V/cm for 1 hr (17). Transfer was-followed by a 30-min incubation in Tris-buffered saline (TBS; 10 mM Tris HCI, pH 7.5/150 mM NaCl) containing 2% (wt/vol) nonfat dry milk powder. Blots were incubated with antibodies for 16 hr at 40C and washed successively with TBS, TBS containing 0.1% Nonidet P-40, and TBS. Blots incubated with monoclonal antibodies were further incubated with goat anti-mouse immunoglobulin conjugated to alkaline phosphatase as described by Darr et al. (13)

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A Nicotiana plumbaginifolia genomic library in the phage Charon 34 was used to isolate and characterize 7 full-length genes and part of an 8th gene encoding chlorophyll a/b-binding (CAB) polypeptides. These genes are arranged in two clusters. All the genes within the clusters are arranged in opposite orientation to their neighbours. The nucleotide sequences of two genes, one from each cluster, show that both genes, designated Cab-E and Cab-C, encode very similar proteins (95.9% of homology) corresponding to type I photosystem II polypeptides. Southern blot analysis suggests that at least 19 CAB genes encoding type I PSII CAB polypeptides are present in the N. plumbaginifolia genome. We also describe the presence within the N. plumbaginifolia genome of CAB genes encoding PSII type II CAB polypeptides and PSI type I CAB polypeptides. The sequences of the 5' flanking region of three different CAB genes (Cab-E, Cab-C, and CAB-F) were determined. Two of them (Cab-C and Cab-F) share extensive homology, whereas the Cab-E promoter shows homology to Cab-C and Cab-F only in a unique region extending from the CAAT box to the TATA box. This conserved sequence is also found in the same position in promoters of CAB genes encoding type I PSII polypeptides from other plant species.

When 14C-labeled chloramphenicol was added to chloramphenicol-producing cultures of Streptomyces strain 13s, radioactive p-nitrophenylserinol, N-acetyl-p-nitrophenylserinol, p-nitrobenzyl alcohol, and p-nitrobenzoic acid were formed. The rate of catabolism varied with culture conditions. It was fastest during rapid growth on a medium supporting low chloramphenicol production. Periodic analysis of cultures indicated that p-nitrophenylserinol accumulated only transitorily and was rapidly N-acetylated. In support of this conclusion 14C-labeled p-nitrophenylserinol was rapidly and completely metabolized, giving mainly the N-acetyl derivative. p-Nitrobenzyl alcohol and p-nitrobenzoic acid were formed in only small amounts during catabolism of chloramphenicol or p-nitrophenylserinol. Chloramphenicol inhibited growth of Streptomyces strain 13s by increasing the lag phase. It also appeared to inhibit antibiotic synthesis. However, cultures supplemented with D-glucose-U-I4C produced radioactive chloramphenicol even when exposed to high concentrations of the antibiotic, and the titer at any time thus represents the product of both catabolic and biosynthetic reactions.

We report here the isolation and nucleotide sequence of a cDNA clone encoding a phtosystem I polypeptide that is recognized by a polyclonal antibody prepared against subunit II of the photosystem I reaction center. The transit peptide processing site was determined to occur after Met50 by N terminal sequencing. The decuced sequence of this protein predicts that the polypeptide has a net positive charge (pI=9.6) and no membrane spanning regions are evident from the hydropathy plot. Based on these considerations and the fact that subunit II is solubilized by alkali treatment of thylakoids, we concluded that subunit II is an extrinsic membrane protein. The absence of hydrophobic regions characteristic of thylakoid transfer domains furthermore implies that subunit II is localized on the stromal side of the membrane.