Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Die prim?re Proteinstruktur von St?mmen des Tabakmosaikvirus

Summary In contrast to chemically induced mutants of tobacco mosaic virus (TMV) in which we have found replacement of one or at most of two amino acids per coat protein chain, the protein chains of naturally occurring TMV strains differ from each other in numerous positions. The complete amino acid sequence of the naturally occurring TMV straindahlemense isolated byMelchers (1940) has been determined. It differs in 30 of the 158 amino acid positions from the TMV wild strainvulgare (Fig. 1). This is the first case in which complete amino acid sequences of the coat proteins of two virus strains can be compared. Such a comparison permits conclusions about the structure of the protein subunits and about certain aspects of the genetic code to be drawn.The electrophoretic mobility curves for the virus rods and the A proteins ofvulgare anddahlemense (Fig. 4) can be explained on the basis of the amino acid sequences of the two strains. Spatial distribution of the positive and negative groups within the protein subunits are discussed. One particular segment of the protein chain appears to be so important for the secondary and/or tertiary structure of the protein subunit that amino acid replacements within this segment in general lead to a loss of infectivity.The 46 cases in which we have exactly located the positions of amino acid differences betweenvulgare and various TMV mutants and strains are summarized in Table 1. Combination of the data in Table 1 with the base compositions of the triplets as obtained from the cell free system ofE. coli permits conclusions about the nucleotide sequence within the triplets to be drawn. The triplets shown in Table 2 represent, at present, the best agreement between the data from the cell free system and the work with TMV mutants.

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Mit 4 Textabbildungen     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

The N-terminal sequence of three coat proteins of foot-and-mouth disease virus.

The N-terminal section of the amino acids sequences of three coat proteins of foot-and-mouth disease virus were determined and compared with the first ten amino acids of the corresponding Mengo virus proteins. In the proteins of the two viruses beginning with aspartic acid, five of ten amino acids are in identical positions.     

A comparison of two phage coat protein-RNA interactions.

The interaction between the coat protein of the group I bacteriophage fr with its translational operator site is compared with the previously studied R17 interaction. The sequence of the two RNA binding sites differ by 2 of 20 nucleotides and two coat proteins by 17 of 129 amino acids. An analysis of the binding of fr coat protein to 24 operator variants revealed that the two proteins recognize operator sequences in virtually the same way. However, fr coat protein binds to nearly every RNA 6 to 14-fold tighter than R17 coat protein. Since the fr operator is a weaker binding variant and the fr coat protein shows a different temperature dependence of binding, it is unlikely that the two systems have different Kas in vivo. RNA fragments containing the operator sequences can initiate the capsid assembly with both fr and R17 coat protein. Surprisingly, the two coat proteins can form a mixed capsid in vitro.     

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus

Summary The amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 M_r polypeptide of CLV-1 (3) and the 40 285 M_r polypeptide of TGMV-A (4) show extensive homologies with the 17 768 M_r and 31 388 M_r polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).     

Amino acid sequence of the coat protein subunit in satellite tobacco necrosis virus

The primary structure of the coat protein subunit in satellite tobacco necrosis virus has been investigated. The results obtained are consistent with and support the proposal for the amino acid sequence made from the nucleotide sequence of RNA (Ysebaert et al., 1980). This would imply that no intervening sequences of RNA occur in the cistron for the satellite tobacco necrosis virus coat protein. The polypeptide chain of the protein consists of 195 amino acid residues. It contains one sulfhydryl group but no disulfide bridges. The distribution of various kinds of amino acid residues along the chain is markedly uneven.     

Genetically engineered mutants of the envelope protein of the RNA-containing bacteriophage

Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.     

Mapping of viral genomic regions important in cross-protection between strains of a potyvirus

Cross-protection was tested between potato and tobacco strains of Potato virus A, a member of the genus Potyvirus (PVA), in tobacco plants. Cross-protection was effective only at the initiation of infection. The potato strains provided only weak cross-protection against the tobacco strain, whereas the tobacco strain provided strong cross-protection against potato strains. The tamarillo strain (TamMV) showed cross-protection phenotypes mostly resembling those of the potato strains. Chimera of the PVA strains were utilized to map viral genomic regions important for cross-protection. The coat protein (CP) encoding region and the helper component proteinase (HCpro) affected cross-protection and virus accumulation. An amino acid substitution at the CP N-terminus reduced virus accumulation and the ability to overcome cross-protection, whereas amino acid substitutions introduced to the HCpro increased virus accumulation and the ability to overcome cross-protection. Closer sequence relatedness between the protector and challenger isolate, as determined by the CP-encoding sequence, was correlated with an increased cross-protection ability. Cross-protection was not overcome by inoculation with nonencapsidated viral RNA. Thus, the differences in cross-protection abilities between PVA strains and chimera were not explained with the "re-encapsidation model" described for strains of Tobacco mosaic tobamovirus but may be associated with a virus infection-induced RNA silencing mechanism.     

Sequence analysis of coat protein and molecular profiling of sunflower necrosis virus (SNV) strains from Indian subcontinent

Sunflower is one of the leading edible oilseed crops of the world and is an important oil-producing crop of India. The sunflower necrosis disease caused by sunflower necrosis virus (SNV) has become a major hurdle for cultivation of sunflower in India. However, there is lack of genetic information and  standard methods for detection and identification of the SNV. To address this issue, we have developed an application using coat protein (CP) to perform molecular profiling of SNV strains. The nucleic acid and amino acid sequence analysis of CP of SNV strains collected from different regions of Maharashtra and Karnataka showed high percent homology (96.89–98.87%). However, 3D structural analysis generated eleven distinct groups of SNV strains.  Comparative bioinformatic analyses of nucleic acid and amino acid sequences with different genera of positive stranded (+) ssRNA viruses established their phylogentic relationship with ~25 (+) ssRNA viruses viz., Ilarvirus, Bromovirus, Cucumovirus, Alfamovirus, Comovirus, Nepovirus, Sequivirus, Potyvirus and Closterovirus. Additionally, the phylogenetic analysis revealed three distinct clusters, wherein major cluster I comprised SNV strains and Tobacco streak virus together showing 99% sequence homology and established closer phylogenetic relationship among all member viruses.     

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein.

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine-and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl-carboxylate pairs are discussed.     

Amino acid sequence of cinnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicein

The phytopathogenic fungi Phytophthora cinnamomi cause systemic leaf necrosis on its non-host tobacco; in culture, it secretes a protein, called cinnamomin, which elicits leaf necrosis and protects tobacco against the pathogen Phytophthora nicotianoe, in a way similar to cryptogein and different from capsicein, elicitins of known amino acid sequences. The cinnamomin sequence has been determined and compared to other elicitins. The differences in the 3 elicitin sequences were correlated to the biological activities: 2 lysines were ascribed as the key amino acids involved in the differential control of protection with respect to necrosis.     

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164     

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7629 nueleotides encoding a polyprotein with 2407 amino acids, from which seven putative proteins may be produced by an autolytie cleavage processing besides the viral coat protein. The RNA2 is 3639 nueleotides and codes for a polypretein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75 % respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.     

cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr≈26 000)

Summary Pea albumin 2 (PA2:M_r26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length.The combined sequence data, together with a comparison of PA2-related polypeptides produced in vitro and in vivo, indicate that PA2 is synthesized without a signal sequence and does not undergo significant post-translational modification. Although both forms of PA2 contain Asn-X-Thr consensus sequences, neither form is glycosylated. Accumulation of PA2 contributes approximately 11% of the sulfur-amino acids in pea seeds (cysteine plus methionine equals 2.6 residues percent). Suppression of levels of PA2 polypeptides and their mRNAs in developing seeds of sulfur-deficient plants is less marked than that for legumin, in spite of the lower content of sulfur-amino acids in legumin.