Phosphopeptides in highly purified calf thymus DNA

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.     

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.     

Evidence for covalent binding between phosphopeptides and terminal deoxynucleotides in highly purified calf thymus DNA

Summary Highly purified DNA from calf thymus nuclei (N-DNA) was found to cleave after reaction with a chelating agent and subsequent dialysis. During the cleavage phosphopeptides (PPs) were released into the dialysates. At the end of the cleavage, approximately one half of the PP material remained with the DNA. Since it was so strongly bound, it was considered to be retained in the DNA structure by covalent bonding. In order to confirm this, a commercial DNA (S-DNA) was ultrasonicated and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. DEAE Sephacel chromatography of the digested material yielded 5 fractions. The fraction 2, having the highest proportion of proteinaceous material, was digested with Pronase. Amino acid analysis of the hydrolysis mixture yielded phosphoserine (Pser), asp, thr, ser, glu, gly, ala, val, ile, leu, and arg. The mixture was chromatographed again on DEAE Sephacel. From this a single fraction, number 5, was found to contain both deoxynucleotides and the amino acids, Pser, asp, ser, glu, and gly in a molar ratio of > 7:3:2:2:5. The mixture obtained by hydrolysis of this fraction with snake venom diesterase was again chromatographed on DEAE Sephacel. This fractionation gave two main peaks, one corresponding to the same 5 amino acids and the other to deoxynucleotide material. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between the deoxynucleotide and amino acid portions.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Multiple forms of rat dentin phosphoproteins

Previous studies have shown that the phosphoprotein from rat dentin is heterogenous and can be partially separated into two fractions by ion-exchange chromatography. These proteins were further characterized by polyacrylamide gel electrophoresis, gel chromatography, and amino acid and phosphate analysis, after chromatographic separations on ion-exchange columns. On 5-15% gradient gels, the phosphoproteins extracted from rat dentin and precipitated by CaCl2 gave three Alcian blue-staining bands with apparent molecular weights in the 90-95,000 range. The two slower-moving bands corresponded to highly phosphorylated proteins (HP) that had phosphoserine contents of greater than 400 residues per thousand and contained little or no valine, leucine, phenylalanine, or arginine. The faster-moving band corresponded to a moderately phosphorylated protein that contained about 250 residues per thousand of phosphoserine and greater quantities of glutamic acid, proline, and several other amino acids than HP. The nature of the phosphoproteins in HP was further studied after total removal of the phosphate with an insoluble form of bovine intestinal alkaline phosphatase. The dephosphorylated product (dP-HP) gave a single major band on gel electrophoresis but showed evidence for two closely related NH2-terminal sequences, Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. The dephosphorylated material was separated into two components (dP-HP1 and dP-HP2) by chromatography on QAE-Sephadex A-25. The amino acid compositions of the two components showed that they differed in their primary structures. This conclusion was verified by the finding of the proline-containing sequence in dP-HP2. In addition to these two groups of phosphoproteins, a third class, LP, contains low levels of phosphoserine and high amounts of glutamic acid (W.T. Butler, M. Bhown, M.T. DiMuzio, and A. Linde, (1981) Coll. Res. 1, 187-199).     

Structure and phosphorylation of the Fujinami sarcoma virus gene product

The Fujinami avian sarcoma virus (FSV) transforming gene product, P140, is a fusion protein which contains both gag-related and FSV-specific methionine-containing tryptic peptides. The virion protease p15 cleaved p140 into two fragments: an N-terminal 33K fragment which contained all but one of the gag-related tryptic peptides and a C-terminal 120K fragment which contained all of the FSV-specific tryptic peptides. The 33K gag-related fragment from P140 phosphorylated in FSV-transformed cells contained only phosphoserine, whereas the 120K C-terminal FSV-specific fragments contained both phosphoserine and phosphotyrosine. P140 isolated from cells infected at the nonpermissive temperature with an isolate of FSV which is temperature sensitive for transformation had a normally phosphorylated 33K fragment, but a hypophosphorylated 120K fragment deficient in both phosphotyrosine and phosphoserine. When P140 was immunoprecipitated from cells and phosphorylated in vitro at tyrosine residues in the immune complex kinase reaction, only the FSV-specific fragment was labeled. These data define the structure of FSV P140 and locate the phosphorylated amino acids within the two regions of the polypeptide.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA.

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Molecular analysis of virulent determinants of enterovirus 71

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5'-NTR and 3'-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (Gly(P710)/Gln(P710)/Arg(P710) and Glu(P729)) on the DE and EF loop of VP1, one (Lys(P930)) on the surface of protease 2A and four nucleotides on three positions (G(P272), U(P488) and A(P700)/U(P700)) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the A(P700)/U(P700) was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.     

Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72.

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

Nucleotide sequence of the 5' terminus of satellite tobacco necrosis virus ribonucleic acid.

Treatment of the RNA of satellite tobacco necrosis virus (STNV) with phosphomonoesterase followed by heat denaturation and treatment with polynucleotide kinase in the presence of [gamma-32P]ATP yields a STNV [5'-32P]RNA containing a homogeneous 5' terminus. Analyses of this STNV [5'-32P]RNA yield the sequence of the first 42 nucleotides from the 5'terminus of STNV RNA. This nucleotide sequence contains the translation initiation AUG codon starting at position 30 from the 5' terminus as indicated by match of subsequent nucleotides with the genetic code assignments for the N-terminal amino acids of STNV coat protein in the 5'-terminal sequence ppAGUAAAGACAGGAAACUU-UACUGACUAACAUGGCAAAACAAC. An interesting feature of this sequence is its potential to form a hairpin loop structure involving perfect Watson-Crick base pairing between the first seven nucleotides and nucleotides at positions 16--22.     

Glycinin A5A4B3 mRNA: cDNA cloning and nucleotide sequencing of a splitting storage protein subunit of soybean

The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5'-terminal non-translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3'-terminal non-translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full-sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post-translational processing to generate an intermediary subunit complex (A5-B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non-covalent interactions. From the results obtained by the Chou-Fasman rules we speculated that the two post-translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.     

Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.     

Cloning and sequence analysis of a cDNA for the alpha-globin mRNA of carp, Cyprinus carpio

A cDNA for alpha-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161-170) and its complete nucleotide sequence was determined. The 5' non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an alpha-globin polypeptide consisting of 142 amino acids. The 3' non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp alpha-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

中国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30基质蛋白基因和融合蛋白基因的分子特征分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行基质蛋白(M)和融合蛋白(F)基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出M和F基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。China/Tib/Gej/07-30的M基因由1483个核苷酸组成,编码335个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为92.4%～97.7%和97.0%～98.2%。F基因由2411个核苷酸组成,编码546个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为85.5%～96.1%和94.3%～98.2%。China/Tib/Gej/07-30的F蛋白含有信号肽序列和跨膜结构域,序列高度变异。F蛋白第104～108位和第109～133位氨基酸位点分别是高度保守的裂解位点和融合肽结构域。F蛋白还含有序列高度保守的三个七肽重复区。China/Tib/Gej/07-30的M基因3′端的非编码区(UTR)长度为443个核苷酸,GC含量高达68.4%,与其他PPRV毒株的同源性为82.4%～93.5%。China/Tib/Gej/07-30的F基因5′UTR区长度为634个核苷酸,GC含量高达70.0%,与其他PPRV毒株序列相似性为76.2%～91.7%。     

The nucleotide sequence of the glucoamylase gene GLA1 from Saccharomycopsis fibuligera KZ

The nucleotide sequence of the 2544-bp PstI fragment carrying the glucoamylase gene of Saccharomycopsis fibuligera KZ, designated as GLA1, has been determined. When compared with the nucleotide sequence of the GLU1 gene one nucleotide substitution was found in the 321- bp of the 5'-flanking region: 24 nucleotides were altered within the 1557 bp of the structural gene causing the deduced protein products of both genes to differ in three amino acids in the signal-peptide region and in eight amino acids of the mature protein. Six nucleotide insertions and 27 substitutions were in the 663 bp of the 3'-flanking region. The gene product expressed and secreted in Saccharomyces cerevisiae into the functional enzyme was not homogeneous. In situ detection of the enzyme in a polyacrylamide gel revealed two dominant and three minor bands.     

Cloning and sequence analysis of a cDNA for the α-globin mRNA of carp, Cyprinus carpio

A cDNA for α-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161–170) and its complete nucleotide sequence was determined. The 5′ non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an α-globin polypeptide consisting of 142 amino acids. The 3′ non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp α-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.