Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis

Plants need to adapt to various stress factors originating from the environment. Signal transduction pathways connecting the recognition of environmental cues and the initiation of appropriate downstream responses in plants often involve intracellular Ca²⁺ concentration changes. These changes must be deciphered into specific cellular signals. Calmodulin-like proteins, CMLs, act as Ca²⁺ sensors in plants and are known to be involved in various stress reactions. Here, we show that in Arabidopsis 2 different CMLs, AtCML37 and AtCML42 are antagonistically involved in drought stress response. Whereas a CML37 knock-out line, cml37, was highly susceptible to drought stress, CML42 knockout line, cml42, showed no obvious effect compared to wild type (WT) plants. Accordingly, the analysis of the phytohormone abscisic acid (ABA) revealed a significant reduction of ABA upon drought stress in cml37 plants, while in cml42 plants an increase of ABA was detected. Summarizing, our results show that both CML37 and CML42 are involved in drought stress response but show antagonistic effects.     

A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery

The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli. This conclusion is based on the finding that mutations altering the prlA gene product restore export of envelope proteins with defective signal sequences. Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins. Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon. Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids. Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

Differential regulation of Dk and Kk major histocompatibility complex class I proteins on the cell surface after infection of murine cells by pseudorabies virus.

After pseudorabies virus (PRV) infection of murine L929 cells, the cell surface expression of major histocompatibility complex (MHC) class I proteins changes such that the total amount of MHC class I molecules remains relatively constant but the levels of the individual alleles Dk and Kk vary. This is an active process involving at least three PRV gene products that act in an allele-specific manner such that cell surface expression of MHC class I Dk is decreased and that of Kk is increased. Our results indicate that an early gene product mediates the overall reduction in Dk protein and a late gene product which is mutant in the attenuated PRV strain Bartha mediates the increase in Kk protein. We provide additional evidence for a third gene product involved in the regulation of the synthesis of both the Dk and Kk proteins. In addition, we show that the early decrease in the Dk protein is not due to a block in synthesis or processing of the complex through the secretory system.     

Reconstitution of nucleotide excision nuclease with UvrA and UvrB proteins from Escherichia coli and UvrC protein from Bacillus subtilis

Recently, an open reading frame which has a deduced amino acid sequence that shows 38% homology to Escherichia coli UvrC protein was found upstream of the aspartokinase II gene (ask) in Bacillus subtilis (Chen, N.-Y., Zhang, J.-J., and Paulus, H. (1989) J. Gen. Microbiol. 135, 2931-2940). We found that plasmids containing this open reading frame complement the uvrC mutations in E. coli. We joined the open reading frame to a tac promoter to amplify the gene product in E. coli and purified the protein to near homogeneity. The apparent molecular weight of the gene product is 69,000, which is consistent with the calculated molecular weight of 69,378 fro the deduced gene product of the open reading frame. The purified gene product causes the nicking of DNA at the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to a thymine dimer when mixed with E. coli UvrA and UvrB proteins and a DNA substrate containing a uniquely located thymine dimer. We conclude that the gene product of the open reading frame is the B. subtilis UvrC protein. Our results suggest that the B. subtilis nucleotide excision repair system is quite similar to that of E. coli. Furthermore, complementation of the UvrA and UvrB proteins from a Gram-negative bacterium with the UvrC protein of Gram-positive B. subtilis indicates a significant evolutionary conservation of the nucleotide excision repair system.     

Transport of hemolysin by Escherichia coli

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.     

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.     

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunological techniques, we have compared the synthesis of the phoA protein (alkaline phosphatase) and the phoS protein (phosphate-binding protein) in response to the level of phosphate in the medium in different genetic backgrounds containing the known alkaline phosphatase control mutations. Both proteins are produced in excess phosphate media in a phoR1a- strain, whereas neither protein is produced in a phoB- strain even under derepression conditions. In four different phoR1c- strains, however, the phoA product cannot be detected in extracts of cells obtained from any growth condition, whereas the phoS product is produced in both excess and limiting phosphate media. It is not yet known if phoR1c- mutants are a special class of mutations within the phoB gene or whether they occur in a separate cistron involved in alkaline phosphatase regulation. From these results we conclude that the expression of the phoA gene is not always co-regulated with expression of the phoS gene product. We have determined that the phoS protein is a component of periplasmic protein band P4 described by Morris et al. (1974). The phoS product lacks sulfur-containing amino acids and is extractable by treatment with polymyxin sulfate. The other component of band P4 contains methionine and/or cysteine and is not extracted by polymyxin sulfate treatment. Like the phoS and phoA proteins, its synthesis is sensitive to the concentration of phosphate in the growth medium. In addition, the existence of a new class of periplasmic proteins synthesized at maximum rate in high phosphate media is demonstrated.     

The Arabidopsis calmodulin‐like protein,CML39, functions during early seedling establishment

During Ca²⁺ signal transduction, Ca²⁺‐binding proteins known as Ca²⁺ sensors function to decode stimulus‐specific Ca²⁺ signals into downstream responses. Plants possess extended families of unique Ca²⁺ sensors termed calmodulin‐like proteins (CMLs) whose cellular roles are not well understood. CML39 encodes a predicted Ca²⁺ sensor whose expression is strongly increased in response to diverse external stimuli. In the present study, we explored the biochemical properties of recombinant CML39, and used a reverse genetics approach to investigate its physiological role. Our data indicate that Ca²⁺ binding by CML39 induces a conformational change in the protein that results in an increase in exposed‐surface hydrophobicity, a property that is consistent with its predicted function as a Ca²⁺ sensor. Loss‐of‐function cml39 mutants resemble wild‐type plants under normal growth conditions but exhibit persistent arrest at the seedling stage if grown in the absence of sucrose or other metabolizable carbon sources. Under short‐day conditions, cml39 mutants display increased sucrose‐induced hypocotyl elongation. When grown in the dark, cml39 mutants show impaired hypocotyl elongation in the absence of sucrose. Promoter–reporter data indicate that CML39 expression is prominent in the apical hook in dark‐grown seedlings. Collectively, our data suggest that CML39 functions in Arabidopsis as a Ca²⁺ sensor that plays an important role in the transduction of light signals that promote seedling establishment.     

Identification of a purC gene from Streptococcus pneumoniae.

A gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase was identified in Streptococcus pneumoniae as a 708-bp segment of the genome encoding a 27,001-Da protein with strong similarity to known PurC proteins. The S. pneumoniae purC gene, found immediately adjacent to the competence induction genes, comAB, was cloned and sequenced. The predicted protein product of purC displayed substantial (> 40%) identity to the entire sequence of the PurC proteins of Bacillus subtilis and Escherichia coli. Function of the S. pneumoniae gene product was demonstrated by complementation of E. coli purC mutations.     

Nucleotide sequence of the Streptococcus pneumoniae hexB mismatch repair gene: homology of HexB to MutL of Salmonella typhimurium and to PMS1 of Saccharomyces cerevisiae.

The Hex mismatch repair system of Streptococcus pneumoniae acts both during transformation (a recombination process that directly produces heteroduplex DNA) to correct donor strands and after DNA replication to remove misincorporated nucleotides. The hexB gene product is one of at least two proteins required for mismatch repair in this organism. The nucleotide sequence of a 2.7-kilobase segment from the S. pneumoniae chromosome that includes the 1.95-kilobase hexB gene was determined. The gene encodes a 73.5-kilodalton protein (649 residues). The spontaneous hex Rx chromosomal mutant allele with which a mutator phenotype has been associated is shown to result from a single base substitution (TAC to TAA) leading to a truncated HexB polypeptide (484 residues). The HexB protein is homologous to the MutL protein, which is required for methyl-directed mismatch repair in Salmonella typhimurium and Escherichia coli, and to the PMS1 gene product, which is likely to be involved in a mismatch correction system in Saccharomyces cerevisiae. The conservation of HexB-like proteins among procaryotic and eucaryotic organisms indicates that these proteins play an important common role in the repair process. This finding also suggests that the Hex, Mut, and PMS systems evolved from a common ancestor and that functionally similar mismatch repair systems could be widespread among procaryotic as well as eucaryotic organisms.     

The plant biotin synthase reaction. Identification and characterization of essential mitochondrial accessory protein components

In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.     

Rye photosystem II. Nucleotide sequence of the psbC gene coding 43- kDa chlorophyll(a)-binding proteins

The structure of the rye chloroplast DNA, which contains psbC gene coding for 43-kDa chlorophyll(a)-binding subunit of photosystem II, is determined. The sequence of trnS (UGA) gene encoding tRNA Ser is located at a distance of 140 bp downstream from the stop codon of psbC gene on the opposite DNA strand. The 5'-terminal part of psbC gene, like in other plants, overlaps by 50 bp the 3'-terminal region of psbD gene coding for D2 protein of photosystem II. The amino acid sequence of the psbC gene product reveals common features with the structure of the psbB gene product (CPa-1 protein). The structural similarity of these two proteins seems to reflect their similar functions.