The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of BaciUus subtilis for use in the positive selection and isolation of insertion sequence (IS)Y'elements in gram-negative bacteria. Cells containing pUCD80 do not grow on medium containing 5% sucrose tinless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NTlRE by selection for growth on sucrose medium. This putative IS element'appears to be unique to Agrobacterium strains.

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cationtransporting ATPases. The corresponding genomic done was isolated and the complete coding sequence was deduced after identification and mapping of introns. Ca2+-ATPases from animals are among the best studied P-type ATPases. Their function is to establish steep Ca2+ gradients across cellular membranes and maintain cytoplasmic Ca2+ at submicromolar concentrations. This allows transient increases in cytoplasmic Ca2+ to mediate the transduction of various signals. These enzymes fall into two categories: Ca2+-ATPases located in the plasma membrane (PM Ca2+-ATPases) and Ca2+-ATPases located in the sarcoplasmic/endoplasmic reticulum membranes (SER Ca2+-ATPases). Primary structure similarity between these two types of enzyme is -30%, whereas similarity within these types is >50%o (8).We have cloned a P-type ATPase unique in both its subcellular localization and its structure. It is found in the plant plastid envelope, a membrane not previously known to contain a P-type ATPase. Its deduced primary structure most closely resembles that of mammalian PM Ca2+-ATPases. It is, however, clearly distinct from the mammalian polypeptides and appears to represent an unusual type of calcium transporter. MATERIALS AND METHODScDNA Cloning. An Arabidopsis thaliana (L. cv. Columbia) shoot cDNA library in AYES (9) was screened with an antiserum against spinach chloroplast envelope proteins in the 55-to 75-kDa size range, and positive clones were isolated and characterized as described (10). This resulted in the cloning and identification of PEAJa (see Fig. 1). Subsequent isolation ofa longer cDNA, PEA]b (see Fig. 1

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.