Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASK), ASKetha (ASK) and ASKiota (ASK). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASK is expressed in the whole embryo during its development, ASK expression is limited to the suspensor cells. No signal was detected for ASK, ASK and ASK in developing embryos.     

Backbone dynamics of the cytotoxic ribonuclease alpha-sarcin by 15N NMR relaxation methods

The cytotoxic ribonuclease -sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of -sarcin through measurement of longitudinal relaxation rates R₁, off resonance rotating frame relaxation rates R₁, and the ¹⁵N¹HNOE of the backbone amide ¹⁵N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. -Sarcin behaves as an axial symmetric rotor of the prolate type (D/D=1.16 ± 0.02) which tumbles with a correlation time _m of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that -sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of -sarcin in the process of recognition of its ribosomal target.     

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

Detection of likely transmembrane β-strand regions in sequences of mitochondrial pore proteins using the Gibbs sampler

The mitochondrial channel VDAC is presumed to fold as a -barrel although the number and identity of transmembrane -strands in the protein are controversial. Previously, a novel multiple alignment algorithm called the Gibbs sampler was used to detect a residue-frequency motif in sequences of bacterial outer-membrane proteins that corresponds to transmembrane -strands in bacterial porins of known structure (Neuwaldet al., 1995,Protein Science,4, 1618. In the present study, this bacterial motif has been used to screen sets of mitochondrial membrane protein sequences, with matches occurring in only two classes of proteins: VDACs and the outer-membrane protein import pore (ISP42, MOM38). These results suggest a structural (and perhaps evolutionary) relatedness between the bacterial and mitochondrial pore proteins, with the mitochondrial subsequences that match the bacterial motif corresponding to transmembrane -strands as in the porins.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Cryptomonad biliproteins — an evolutionary perspective

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as 50000 kDa '₂ complexes which carry one bilin on the and three on the subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The subunit is encoded on the chloroplast genome, whereas the subunits are encoded by a small nuclear multigene family. The subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is primitive and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein and subunits.Abbreviations Chl chlorophyll - CER chloroplast endoplasmic reticulum - SSU rRNA small subunit ribosomal RNA     

On the heterogeneity of the slow reassociating (“unique”) DNA

Summary The slow reassociating fraction of mouse DNA (unique DNA), when allowed to reassociate in 0.14 m sodium phosphate buffer at 50 °C showed a biphasic melting curve with a transition at 78–80 °C. On the basis of this feature, the slow reassociating DNA was separated preparatively into two fractions: unique DNA I and II. Their duplexes showed differences with respect to thermal stability, S1 nuclease resistance and rate of reassociation. About one third of the sequences in each fraction were fraction-specific. The conclusion was drawn that for unique DNA I these should be the low repetitive or single copy related sequences (multigene families) and for unique DNA II—the unrelated single copy sequences or recent families of low repetitive not yet diverged sequences.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

Purification and properties of a glycerophosphocholine phosphodiesterase from bovine brain myelin

An enzyme releasing phosphocholine from glycerophosphocholine was purified to apparent homogeneity based upon SDS-PAGE. The enzyme was liberated from lyophilized bovine myelin by differential detergent extraction and final purification was accomplished with Q-Sepharose Fast Flow chromatography yielding an apparently homogenous protein. The molecular mass based upon PAGE was approximately 14 kDa. The enzyme was also capable of releasing p-nitrophenol from p-nitrophenyl-phosphocholine. Maximal activity was obtained with 0.2 mM ZnCl₂ or 1 mM CoCl₂. p-Nitrophenylphosphocholine and phosphocholine were competitive inhibitors of glycerophosphocholine hydrolysis with Ki's of 0.028 mM and 0.03 mM respectively. Glycerophosphocholine and phosphocholine were competitive inhibitors of p-nitrophenylphosphocholine hydrolysis with Ki's of 0.5 mM and 1.75 mM respectively.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - GPC glycerophosphocholine - pNPPC p-nitrophenylphosphocholine - OG octyl--glucoside - PMSF phenylmethylsulfonylfluoride - CNPase 23-cyclic nucleotide 3-phosphodiesterase     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

Evolutionary implications of error amplification in the self-replicating and protein-synthesizing machinery

Summary Evolutionary constraints operating on animal mitochondrial tRNA were estimated to be reduced to about 1/30 of those that apply to cytoplasmic tRNA. In the nuclear-cytoplasmic system, an effect of a mutation tRNA is likely to be amplified through positive feedback loops consisting of DNA polymerases, RNA polymerases, ribosomal proteins, aminoacyl-tRNA synthetases, tRNA processing enzymes, and others. This amplification phenomenon is called an error cascade and the loops that cause it are called error loops. The freedom of evolutionary change of cytoplasmic tRNA is expected to be severely restricted to avoid the error cascade. In fact, cytoplasmic tRNA is highly conserved during evolution. On the other hand, in the animal mitochondrial system, all of the proteins involved in error loops are coded for in the nuclear genome and imported from the cytoplasm, and accordingly the system is free from the error cascade. The difference in constraints operating on animal tRNA between cytoplasm and mitochondria is attributed to the presence or absence of error loops. It is shown that the constraints on mitochondrial tRNA in fungi are not as relaxed as those in animals. This observation is attributed to the presence of an error loop in fungal mitochondria, since at least one protein of the mitochondrial ribosome is coded for in the mitochondrial genome of fungi. The evolutionary rates of proteins involved in the processing of genetic information are discussed in relation to the error cascade.A preliminary version of this paper was presented at the International tRNA Workshop (Hakone, Japan, March 1983) and at the Second International Colloquium on Endocytobiology (Tübingen, FRG, April 1983)     

Subunit composition of the zinc proteins alpha- and beta-lipovitellin from chicken

Chicken - and -lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per -lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per -lipovitellin, respectively. The subunits of -lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of - and -lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from - and -lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of - and -Lv, respectively, and two peptides of the 30-kDa subunit of - and -lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of - and -lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of -lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 Å for all backbone atoms and 0.40 Å for the backbone atoms of the central -sheet. The core structure of the SH2 domain contains an antiparallel -sheet flanked by two parallel -helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.