AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca2 + than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.

Plasmid pACYC184 contains an ssi signal for initiation of single-strand phage DNA replication.

Using the plaque assay system for screening the single-strand (ss) initiation determinant (ssi) sequences, we have found that 119-bp region in pACYC184, a derivative of the plasmid P15A of Escherichia coli, can direct such ss DNA initiation. This region is located downstream from the P15A origin of replication and conserves consensus sequences of the ssi signals found in the other plasmids. Signals for ss DNA initiation are defined as nucleotide sequences present on ss DNA templates and required for priming DNA synthesis. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. In this region, we found a potential stem-and-loop structure. The 119-bp DNA segment of plasmid pACYC184 cloned in f1R199 filamentous phage could direct rifampicin-resistant conversion of the ss DNA to the double-stranded replicative form.

Mutational analysis of an ssi region carried by plasmid pACYC184.

To investigate the functional contribution of some structural components of the signal that directs single-stranded initiation of DNA replication (ssi signal) carried by a 119-nt segment of plasmid pACYC184 (Bahk et al., 1988), we constructed mutants carrying one-base substitutions and insertions using oligodeoxyribonucleotide (oligo) directed mutagenesis. Two one-base substitution mutants were obtained. The mutants, M13 delta lac 184/Sp and M13 delta lac 184/Ev, carried an SplI site and an EcoRV site, respectively, created by base substitution. Three kinds of synthetic oligos, that is, a 10-bp EcoRI linker, an 8-bp ScaI linker and an 8-bp SmaI linker, were inserted into the SplI site of M13 delta lac 184/Sp, and into the EcoRV site of M13 delta lac 184/Ev. The SSI activity of each mutant examined indicated that the one-base substitutions had different effects on the SSI functions of the altered ssi signals. This fact suggests that some structural components within the 119-bp region make distinct contributions to the SSI function. Moreover, when the three kinds of synthetic linkers were inserted into the mutants M13 delta lac 184/Sp and M13 delta lac 184/Ev, each of the insertion mutations affected the rate of conversion of ss DNA to RFI in vivo and the growth of the recombinant phages in a distinct manner. Judging from the above results, the base composition and the length of a certain specific site were crucial for maintenance of the SSI functional activity, and structural components of the ssi signal contributed distinctly to the SSI function.

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172.

A DNA fragment (pCHI5422) containing two genes encoding a 54-kD and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 microM (min)-1 mg-1 and 17 microM (min)-1 mg-1 on the natural swollen chitin, respectively.

Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme.

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45 degrees C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 mumol min-1 mg-1 and 60 mumol min-1 mg-1, respectively.

Molecular characterization of a rab-related small GTP binding protein cDNA from rice (Oryza sativa L. IR-36).

To study physiological roles of plant small GTP binding proteins, we isolated a cDNA clone (ORrab2) encoding the rab-related small GTP binding protein from rice (Oryza sativa L. IR-36) by using human cDNA rab2 as a probe. The deduced amino acid sequence of the ORrab2 gene shared all the conserved regions, important for GTPase/GTP binding activities, with those of other small GTP binding proteins. ORrab2 is a 1028 bp long cDNA, encodes a 23.2 kDa protein which shows 85.2% similarity on the amino acid sequence level to the Hrab2 protein, and was used as a probe. Through Southern and Northern blot analyses, we found that ORrab2 is a single copy gene and actively expressed at the stages of cell division and elongation. We investigated GTP binding abilities by a filter assay procedure. Deletion of a binding motif, GDTGVGKS, within an ORrab2 protein showed a significant decrease of GTP binding affinity, suggesting its important role in nucleotide binding.

Cold accumulation of SCOF-1 transcripts is associated with transcriptional activation and mRNA stability.

Cold acclimation enhances the transcription of several cold regulated (COR) genes. However, little is known about whether the elevation of the transcriptional level of the COR genes is due to transcriptional activation, or mRNA stability by a low temperature. Recently, we cloned a novel cold-inducible zinc finger protein gene from soybean, SCOF-1, which may function as a positive regulator of the COR gene expression . Here we report that the elevation of the SCOF-1 transcript level by cold stress is associated with both transcriptional activation and post-transcriptional mRNA stability under a low temperature. A nuclear run-on assay reveals that cold acclimation elevates the SCOF-1 transcript about three-fold compared to that of non-acclimated soybean nuclei. Furthermore, SCOF-1 transcripts increased substantially by a low temperature in transgenic tobacco plants that constitutively expressed SCOF-1 under the control of a constitutive cauliflower mosaic virus (CaMV) 35S promoter. When a transcription inhibitor, cordycepin, was treated with the deacclimating soybean cell, the decay level of the SCOF-1 transcripts was delayed significantly. This suggests that it may affect de novo protein synthesis, which degrades the SCOF-1 mRNA at room temperature. In addition, a secondary structure may be involved in the mRNA stability of SCOF-1 under a low temperature.

A runaway-replication plasmid pSY343 contains two ssi signals.

Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively. We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199. A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A. Van der Ende, R. Teerstra, H. Van der Avoort, and P.J. Weisbeek, 1983, Nucleic Acids Res. 11, 4957-4975) was found, three copies in 170P and one in 93F. These two ssi signals contain possible stem and loop structures. The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region. Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection. The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection.

Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli.

A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography. The molecular mass of RGA1 bearing a His tag was approx. 49 kDa. Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots. It exists at particularly high levels in the immature embryo after pellicle extrusion. In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants. When bound to guanosine 5'-(gamma-thio)triphosphate (GTP[S]), the RGA1 protein was partially protected from tryptic proteolysis. In the presence of GTP[S], trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size. When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion. Photoaffinity labelling with [alpha-32P]GTP and a GTP[S]-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide. Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM). The rate of guanine nucleotide binding of RGA1 (kon,GTP[S]=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2. The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.

Isolation and characterization of three soybean extensin cDNAs.

We have characterized three different soybean (Glycine max) mRNAs that encode apoproteins of extensins, a family of cell wall hydroxyproline-rich glycoproteins (HRGPs). These transcripts encoded distinctive Tyr-rich proteins containing characteristic Ser-Pro4 sequences organized in higher-order repetitive units. The first transcript encoded an extensin SbHRGP-1 containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Val-Tyr-Lys, with Val occasionally replaced by Ile or Tyr. The second transcript encoded the SbHRGP-2 protein containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Tyr-Tyr-Lys/His. The third transcript encoded the SbHRGP-3 protein containing a variant of 9- or 10-amino acid canonical repeats: Ser-Pro4-Tyr-Lys-Tyr-Pro, Ser-Pro5-Tyr-Lys-Tyr-Pro, and Ser-Pro4-Val-Tyr-Lys-Tyr-Lys, respectively. The dramatic amino acid substitutions in the Tyr-rich blocks (Tyr-X-Tyr-Lys) among these HRGPs indicate that each SbHRGP may have a different function in cell wall architecture.

Isolation of two soybean G-box binding factors which interact with a G-box sequence of an auxin-responsive gene.

G-box binding factors (GBFs) constitute a family of plant DNA-binding proteins that bind to the G-box motif, a regulatory cis element present in many plant genes with a palindromic DNA motif of CACGTG. Previously TCCACGTGTC, a G-box motif, from an auxin responsive gene GmAux28 has been identified as a sequence-specific protein-binding site. Here the isolation of two soybean cDNA clones, referred to as SGBF-1 and SGBF-2, encoding proteins which bind to the G-box motif is reported. The primary structure of SGBF-1 and SGBF-2 predicts that these proteins contain a basic leucine zipper (bZIP) DNA-binding domain and an N-terminal proline-rich domain. A dramatic difference in the pattern of protein-DNA complex formation was observed when recombinant SGBF-1 and SGBF-2 proteins were analyzed by electrophoretic mobility shift assays (EMSAs). The SGBF-1 binding pattern obtained with the G-box probe resulted in three major retarded bands while the SGBF-2 formed a single complex. This shows that the characteristically diffuse banding pattern of plant nuclear proteins interacting with the G-box is also observed in a binding assay using only one recombinant GBF. EMSAs were performed with a few selected binding sequences to study the effect of flanking nucleotides to the hexanucleotide G-box core motif. The binding specificity of the SGBF proteins resembles that described for type A cauliflower nuclear G-box binding proteins which bind class I G-box elements [(G/T)(C/A)CACGTG(G/T)(A/C)]. Phylogenetic analysis of 13 GBF-like proteins from various plant species reveals that the SGBF-1 and SGBF-2 proteins belong to different lineages, suggesting that they may have distinct functions in activating transcription.

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Overproduction of indole acetic acid in Azospirillum lipoferum using the Escherichia coli trp operon.

A recombinant plasmid carrying the trp operon from Escherichia coli, which synthesizes tryptophan from chorismate, was constructed by using a broad host range plasmid vector pRK290; a mutant trp plasmid for tryptophan overproduction was then selected. The physiological, biochemical, and genetic properties of the Azospirillum lipoferum KY6, a potential nitrogen fixer of rice, harbouring the recombinant trp plasmid pMJC1 and its mutant pMJC101, were compared with those of the wild-type bacteria. Anthranilate synthetase is known to be the trpE gene product which plays a key role in the regulatory step in the feedback control of tryptophan biosynthesis. The enzyme activity of the Azospirillum lipoferum KY6 carrying pMJC1 or pMJC101 was respectively 7- and 30-fold higher than that of the wild type in the presence of 10(-4)M tryptophan. As expected, the amount of tryptophan biosynthesis in A. lipoferum KY6 (pMJC101) was increased approximately 100-fold as compared with the wild type, which led to overproduction of indole acetic acid even without addition of exogenous tryptophan. Moreover, the recombinant trp plasmid was fairly stable in A. lipoferum KY6 host, showing only 25% loss of the plasmid itself or the trp insert after 40 generations.

Isolation of an additional soybean cDNA encoding Ypt/Rab-related small GTP-binding protein and its functional comparison to Sypt using a yeast ypt1-1 mutant.

We have previously reported the isolation of a gene from a soybean cDNA library encoding a Ypt/Rab-related small GTP-binding protein, Sypt. Here, we report the isolation of a second Ypt/Rab-related gene, designated Srab2, from the same soybean cDNA library. And we compare the in vivo function of the two soybean genes utilizing a yeast ypt1-1 mutant. The Srab2 gene encodes 211 amino acid residues with a molecular mass of 23 169 Da. The deduced amino acid sequence of the Srab2 is closely related to the rat (76%) and human (75%) Rab2 proteins, but it shares relatively little homology to Sypt (46%) and Saccharomyces cerevisiae ypt proteins (41%). Genomic Southern blot analysis using the cDNA insert of Srab2 revealed that it belongs to a multigene family in the soybean genome. The protein encoded by Srab2 gene, when expressed in Escherichia coli, disclosed a GTP-binding activity. The expression pattern of the Srab2 gene is quite different from that of the Sypt gene. The Srab2 gene is predominantly expressed in the plumule region, while expression was very low in the other areas in soybean seedlings. On the other hand, the Sypt mRNA is not detectable in any tissues of soybean seedlings grown in the dark. However, light significantly suppressed the Srab2 gene expression, but enhanced the transcript levels of the Sypt gene in leaf and, at even higher levels, in root tissues. When the Srab2 and Sypt genes are introduced separately into a S cerevisiae defective in vesicular transport function, the Srab2 gene cannot complement the temperature-sensitive yeast ypt1-1 mutation at all, in contrast to the Sypt gene. In conclusion, the difference of functional complementation of the yeast mutation together with differential expression of the two genes suggest that the in vivo roles of the Srab2 and Sypt genes may be different in soybean cells.

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.

The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1.

Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Ras superfamily (G1-G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sar1 family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S. cerevisiae and that they may perform similar biological functions.

Guanine-nucleotide binding and hydrolyzing kinetics of ORrab2, a rice small GTP-binding protein expressed in Escherichia coli.

The ORrab2 gene encodes a GTP-binding protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%), Hrab4 (51.9%), Hrab1 (46.2%), YPT (40.7%), Hrab3B (40.0%), Hrab3A (38.1%), SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatography. The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa. The guanine-nucleotide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [gamma-35S]GTP[S]/mol of protein with a Kd value of 56.83 nM. The ratios k(off GDP)/k(off GTP) of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (k(cat)) of Pi release against [gamma-32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [gamma-32P]GTP (kGTPase) were calculated to be 432 x 10(-4) +/- 8 x 10(-4) min(-1) and 172 x 10(-4) +/- 2 x 10(-4) min(-1), respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4.

Molecular cloning and functional characterization of a cDNA encoding nucleosome assembly protein 1 (NAP-1) from soybean.

NAP-1, a protein first isolated from mammalian cells, can introduce supercoils into relaxed circular DNA in the presence of purified core histones. Based on its in vitro activity, it has been suggested that NAP-1 may be involved in nucleosome assembly in vivo. We isolated a cDNA clone encoding a soybean NAP-1 homolog, SNAP-1. The SNAP-1 cDNA contains an open reading frame of 358 amino acids residues with a calculated molecular weight of 41 kDa. The deduced amino acid sequence of SNAP-1 shares sequence similarity with yeast NAP-1 (38%) and human hNRP (32%). Notable features of the deduced sequence are two extended acidic regions thought to be involved in histone binding. SNAP-1 expressed in Escherichia coli induces supercoiling in relaxed circular DNA, suggesting that SNAP-1 may have nucleosome assembly activity. The specific activity of SNAP-1 is comparable to that of HeLa NAP-1 in an in vitro assay. Western analysis reveals that SNAP-1 is expressed in the immature and young tissues that were examined, while mature tissues such as old leaves and roots, show very little or no expression. NAP-1 homologs also appear to be present in other plant species.

Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus.

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::beta-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/ antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses.