A most primitive primodial gene as a building block of the genes for the adenylate kinase, F1-ATPase subunits (epsilon and gamma), aminoacyl-tRNA synthetase, and core enzyme subunits

Internal homology units of F1-ATPase epsilon and gamma subunits were searched by computer-aided methods. The epsilon in E. coli (EC) and maize chloroplast (Ch1) was found to consist of three homologous domains, named domains I, II and III (amino acids 1-47, 48-95 and 96-139 for EC). The gamma in E. coli was demonstrated to have at least six homologous domains, tentatively named here domains I-III and V-VII (I = aa 1-23, II = 26-69, III = 71-112, V = 150-192, VI = 196-242, VII = 285-329), with leaving a region IV (113-149) unclassified. Adenylate kinases (AK's) in pig and E. coli were found to have three internal homology units, named I, I' and II (I = aa 1-47, I' = 48-79, II = 80-124 for pig). Statistical evaluations and dot matrix analyses at both base and amino acid sequence levels have confirmed that all of these repeating units, being about 46 amino acids long, are homologous with one another. Of these, epsilon III, II, gamma VII and AK II domains were most conservative and some of them showed homology to core enzyme alpha and an internal repeating unit of tryptophanyl-tRNA synthetase (Trp-RS). Thus these homology unit-encoding gene segments must be relics of a primodial gene.     

Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types. Specific expression of the mRNA in skeletal muscle

Two types of calcium-dependent protease with distinct calcium requirements (termed muCANP and mCANP) are known in mammalian tissues. These two isozymes consist of different large (80-kDa) subunits (mu- or m-types) and identical small (30-kDa) subunits. By screening human and rat muscle cDNA libraries with a cDNA probe for the chicken CANP large subunit, which has a structure similar to both the mammalian mu- and m-types, a cDNA clone encoding a novel member of the CANP large subunit family was obtained. The encoded protein (designated "p94") consists of 821 amino acid residues (Mr 94,084) and shows significant sequence homology with both human mu-type (54%) and m-type (51%) large subunits. p94 can be divided into four domains (I-IV) as reported for the CANP large subunit family. Domains II and IV are potential cysteine protease and calcium-binding domains, respectively, and have sequences homologous to the corresponding domains of other CANP large subunits. However, domain I of p94 is significantly different from others. Moreover, p94 contains two unique sequences of 62 and 77 residues in domains II and III, respectively. In contrast to the ubiquitous expression of mu- and m-types, Northern blot analysis revealed that the mRNA for p94 exists only in skeletal muscle with none detected in other tissues including heart muscle and smooth muscles such as intestine.     

Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.     

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.     

Quick Identification of the Members of the Glutamine Synthetase Gene Family from Sunflower by Simultaneous Amplification of cDNA with Degenerate Primers

A single degenerate glutamine synthetase (GS)-specific primer was used to amplify the 3′ end of cDNAs derived from different GS genes that are expressed in leaves and roots of sunflower (Helianthus annuus L. cv. Peredovic). Four types of GS cDNA (I, II, III and IV) were simultaneously amplified from leaves and five types (I, II, V, VI, VII) from roots with a minimum investment of time and experimental work. cDNAs II, III and IV encode chloroplastic isoforms as deduced by the presence of chloroplastic GS-specific features in their sequences. The rest of cDNAs codifies cytosolic isoforms. Using cDNA-specific probes and primers, homologous sequences to all GS cDNAs amplified from cv. Peredovic, except to cDNAs III and IV, were detected in the inbred line R41. This result strongly suggests that the three cDNAs for chloroplastic isoform are allelic sequences from the same locus, and since cDNA type IV contains sequences derived from cDNAs II and III, it indicates a recombinational origin. The results presented are consistent with the existence of a GS gene family in sunflower with at least five members. Four of them, named ggs1.1 to ggs1.4, codify for the cytosolic isoforms (cDNAs I, V, VI and VII). A fifth member, named ggs2, from which three allelic sequences (cDNAs II, III and IV) have been cloned, encodes the chloroplastic isoform. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different extracellular domains of the neural cell adhesion molecule (N- CAM) are involved in different functions

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I- II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig- like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig- like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type III repeats I-II. Intracellular concentrations of Ca2+ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures.     

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.     

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.     

Requirement of the N-terminal region of orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.     

Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 A distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II.III.IV is required, which contrasts the known compact RF crystal structures. The crystal structure of Thermus thermophilus RF2 was determined and compared with solution structure of T. thermophilus and Escherichia coli RF2 by microcalorimetry, circular dichroism spectroscopy and small angle X-ray scattering. The structure of T. thermophilus RF2 in solution at 20 degrees C is predominantly compact like the crystal structure. Thermodynamic analysis point to an initial melting of domain I, which is independent from the melting of the core. The core domains II.III.IV melt cooperatively at the respective physiological temperatures for T. thermophilus and E. coli. Thermodynamic analyses and the X-ray scattering results for T. thermophilus RF2 in solution suggest that the compact conformation of RF2 resembles a physiological state in absence of the ribosome.     

Comparison of the nucleotide sequences at the 3′-terminal region of RNAs from RNA coliphages

In order to study the genealogical relationships among four groups (I to IV) of RNA coliphages, we sequenced 200 to 260 nucleotides from the 3′ termini of 14 phage RNAs according to the method of Sanger et al. (1977), and compared the results. It was found that the sequences of phage RNAs in the same group were extremely homologous (about 90%). On the other hand, when the sequences were compared with those from other groups, they were seen to be only about 50 to 60% homologous between group I and group II, and about 50% homologous between group III and group IV. In other combinations, such as groups I (or II) and III, and groups I (or II) and IV, however, the extent of homology was small. Furthermore, the sequences up to 30 residues from the 3′ end were found to be about 90% homologous between groups I and II, and between groups III and IV.These results confirm our previous findings, that the sequences located in the proximity of the 3′ end of phage RNA in the same group were well-conserved (Inokuchi et al., 1979), and that close relationships exist between groups I and II, and between groups III and IV (Furuse et al., 1979).     

Paramecium calmodulin mutants defective in ion channel regulation can bind calcium and undergo calcium-induced conformational switching

Calmodulin (CaM) is an essential eukaryotic protein that binds calcium ions cooperatively at four EF-hand binding sites to regulate signal transduction pathways. Interactions between the apo domains of vertebrate CaM reduce the calcium affinities of sites I and II below their intrinsic values, allowing sequential opening of the two hydrophobic clefts in CaM. Viable domain-specific mutants of Parameciumcalmodulin (PCaM) differentially affect ion channels and provide a unique opportunity to dissect the roles of the two highly homologous half-molecule domains. Calcium binding induced an increase in the level of ordered secondary structure and a decrease in Stokes radius in these mutants; such changes were identical in direction to those of wild type CaM, but the magnitude depended on the mutation. Calcium titrations monitored by changes in the intrinsic fluorescence of Y138 in site IV showed that the affinities of sites III and IV of wild type PCaM were (i) higher than those of the same sites in rat CaM, (ii) equivalent to those of the same sites in PCaM mutants altered between sites I and II, and (iii) higher than those of PCaM mutants modified in sites III and IV. Thus, calcium saturation drove all mutants to undergo conformational switching in the same direction but not to the same extent as wild type PCaM. The disruption of the allosteric mechanism that is manifest as faulty channel regulation may be explained by altered properties of switching among the 14 possible partially saturated species of PCaM rather than by an inability to adopt two end-state conformations or target interactions similar to those of the wild type protein.     

The complete amino acid sequence of rice bran trypsin inhibitor

The complete amino acid sequence of a double-headed trypsin inhibitor (RBTI) from rice bran was determined by a combination of limited proteolysis of the native inhibitor with Streptomyces griseus trypsin at pH 3 and conventional methods. RBTI consists of 133 amino acid residues including 18 half-cystine residues which are involved in 9 disulfide bridges in the molecule. The limited proteolysis at pH 3 produced a major split of Lys(83)-Met(84) and a minor split of Arg(107)-Val(108) together with a non-enzymatic hydrolysis of Asp(19)-Pro(20) in the molecule. The established sequence showed that RBTI is composed of 4 domains, domains I and III, and domains II and IV being homologous to the first and the second domains of soybean Bowman-Birk inhibitor, respectively, indicating that RBTI has a duplicated structure of the Bowman-Birk type inhibitor.     

Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C

The mRNA levels for four types of inositol phospholipid-specific phospholipase C (PLC) in various tissues and cell cultures have been studied by Northern analysis using cDNA probes for PLC isozyme I, II, and III [Sue, P.-G., Ryu, S.H., Moon, K.H., Sue, H.W., and Rhee, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and Cell 54, 161-169], and the recently identified isozyme IV. All four types are ubiquitously expressed in rat tissues, but the levels of the mRNAs vary among tissues and cell lines. PLC-I mRNA levels are extremely high in brain and rat C6 glioma cells with lower levels in other tissues tested. PLC-II and -III have a more widespread distribution, with relatively high levels in brain, lung, spleen, thymus, and testis in the case of PLC-II, and in skeletal muscle, spleen, and testis for PLC-III. PLC-II and -III mRNAs were also detected in all cell lines examined except human promyelocytic HL60 cells. PLC-IV mRNA levels are extraordinarily high in spleen and HL60 cells. These results indicate that rat C6 glioma cells, together with most rat tissues, contain all four PLC isozymes. Other cultured cell types examined also contain two or three PLC isozymes except for HL60 cells, which contain only PLC-IV. The concomitant expression of PLC isozymes in cultured cells suggests a diverse function for PLC isozymes in single cells.     

Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal.

We have previously shown that most of the defective interfering (DI) RNA of mouse hepatitis virus (MHV) are not packaged into virions. We have now identified, after 21 serial undiluted passages of MHV, a small DI RNA, DIssF, which is efficiently packaged into virions. The DIssF RNA replicated at a high efficiency on its transfection into the helper virus-infected cells. The virus released from the transfected cells interfered strongly with mRNA synthesis and growth of helper virus. cDNA cloning and sequence analysis of DIssF RNA revealed that it is 3.6 kb and consists of sequences derived from five discontinuous regions of the genome of the nondefective virus. The first four regions (domains I to IV) from the 5' end are derived from gene 1, which presumably encodes the RNA polymerase of the nondefective virus. The entire domain I (859 nucleotides) and the first 750 nucleotides of domain II are also present in a previously characterized DI RNA, DIssE, which is not efficiently packaged into virions. Furthermore, the junction between these two domains is identical between the two DI RNAs. The remaining 77 nucleotides at the 3' end of domain II and all of domains III (655 nucleotides) and IV (770 nucleotides) are not present in DIssE RNA. These four domains are derived from gene 1. In contrast, the 3'-most domain (domain V, 447 nucleotides) is derived from the 3' end of the genomic RNA and is also present in DIssE. The comparison of primary sequences and packaging properties between DIsse and DIssF RNAs suggested that domains III and IV and part of the 3' end of domain II contain the packaging signal for MHV RNA. This conclusion was confirmed by inserting these DIssF-unique sequences into a DIssE cDNA construct; the in vitro-transcribed RNA from this hybrid construct was efficiently packaged into virion particles. DIssF RNA also contains an open reading frame, which begins from domain I and ends at the 5'-end 20 bases of domain III. In vitro translation of DIssF RNA and metabolic labeling of the virus-infected cells showed that this open reading frame is indeed translated into a 75-kDa protein. The structures of both DIssE and DIssF RNAs suggest that a protein-encoding capability is a common characteristic of MHV DI RNA.     

The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).     

Molecular dissection of the ST8Sia IV polysialyltransferase. Distinct domains are required for neural cell adhesion molecule recognition and polysialylation

Polysialic acid, a homopolymer of alpha2,8-linked sialic acid expressed on the neural cell adhesion molecule (NCAM), is thought to play critical roles in neural development. Two highly homologous polysialyltransferases, ST8Sia II and ST8Sia IV, which belong to the sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III, which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion mutants. To identify domains responsible for NCAM polysialylation, different segments of the ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the carboxyl-terminal region of ST8Sia IV. However, chimeric enzymes that contain only the carboxyl-terminal segment of ST8Sia IV and the amino-terminal segment of ST8Sia III showed very weak activity toward NCAM, even though they had strong activity in polysialylating themselves. In fact, chimeric enzymes containing the amino-terminal portion of ST8Sia IV fused to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM recognition domain is distinct from the polysialylation domain and that some chimeric enzymes may act as a dominant negative enzyme for NCAM polysialylation.