Structure of three spliced mRNAs from region E3 of adenovirus type 2

A cDNA library representing early adenovirus type 2 (Ad2) mRNA was constructed. The cDNA copies were inserted into the PstI cleavage site of the pBR322 plasmid, and clones containing sequences from region E3 of the Ad2 genome were identified by colony hybridization. Selected clones were characterized by restriction enzyme cleavage, hybridization, and partial DNA sequence analysis. The precise structure of three spliced mRNAs was established by comparing the results with the DNA sequence of region E3 from Ad2 (Herissé et al., Nucl. Acids Res. 8 (1980) 2173--2191; Herissé and Galibert, Nucl. Acids Res. 9 (1981) 1229--1249). One of the characterized mRNA species encodes the E3/19K glycoprotein, whereas the other two most likely encode the E3/14K protein. The results demonstrate, moreover, that certain splice points which are used to generate the major E3 mRNAs are also used to splice the supplementary leader segments to the fibre mRNA at late times after infection. Two separate poly(A)-addition sites were identified in region E3 by analysis of the cDNA clones; one is preceded by the hexanucleotide sequence AAUAAA, whereas the other is preceded by an altered hexanucleotide, having the sequence AUUAAA.     

Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization

The complete nucleotide sequence of hepatitis B virus (HBV) DNA from Dane particles of subtype adr was determined. The 3215-bp sequence showed the presence of genes for the surface antigen (226 amino acids) and core antigen (183 amino acids), in addition to two (long and small) open reading frames (ORFs) capable of coding the 843 and 154 amino acids. These ORFs differed from those of the other adr clones so far reported [Ono et al., Nucl. Acids Res. 11 (1983) 1747–1757; Fujiyama et al., Nucl. Acids Res. 11 (1983) 4601–4610]. The gene organization of HBV DNA was found to be well conserved irrespective of subtype. The direct repeat of the undecanucleotide sequence near the 5′ ends of the short (S) and long (L) strands of HBV DNA and the two small direct repeats between both 5′ ends were found to be characteristic structures.     

Cloning and sequencing of the bovine adenovirus type 2 early region 4

Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characterisitics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5–100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14 kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3–6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.     

Sequences from the beginning of the fiber messenger RNA of adenovirus-2.

Small restriction fragments, from around co-ordinate 86.6 on the adenovirus-2 genome, have been used as primers for direct DNA sequence analysis by Sanger's (Sanger et al., 1977) chain termination method with Ad2² DNA as template. The genomic sequences obtained have been compared with sequences deduced using fiber messenger RNA from Ad2 or Ad2⁺ ND5-infected cells as template. With one primer, Hha 54, the sequences complementary to mRNA match those of the genome for 10 nucleotides but then differ from those found on the genome because this primer hybridizes near the point at which the leader sequence becomes joined to the main body of fiber mRNA. Using Ad2⁺ ND5 fiber mRNA as template, the sequence beyond the point of divergence matches the known sequence of the third leader component of one of the late Ad2 mRNAs, that encoding the hexon polypeptide. With Ad2 fiber mRNA, a heterogeneous sequence continuation is found, in accordance with earlier findings that two major species of fiber mRNA are present, which differ in the nature of the leader component joined to the main body of fiber mRNA (Chow &; Broker, 1978). Nevertheless, the data suggest that both leader components appear to become joined to the same nucleotide at the start of the main body of fiber mRNA.The AUG codon, which probably encodes the N-terminus of the fiber polypeptide, occurs just two nucleotides beyond the point at which these leader segments become spliced to the main body of fiber mRNA.     

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.     

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155–9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [ Science 230 (1985) 1350–1353] and results in a more than 10⁴-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 μg of tumor DNA.     

Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3'' splice-site consensus sequence.

The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, characterizes the genes of nuclear encoded mitochondrial proteins.     

Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Mapping of a 14,000-dalton antigen to early region 4 of the human adenovirus 5 genome.

An early 14,000-dalton (14K) antigen of adenovirus 5, hitherto designated 10.5K and thought to be from early region 1 (E1), has been shown to be a product of region E4 on the following evidence. In KB cells infected with the adenovirus 5 mutants dl312 and dl313, containing large deletions in region E1, this antigen was produced in a form having the same mobility as that in wild-type infections. In a range of rodent cells transformed by adenovirus 5 DNA, the synthesis of 14K antigen and the ability of these cells to elicit an immune response to this protein both correlated with the presence of sequences from region E4 of the viral genome. A 14K polypeptide was synthesized in a cell-free system programmed with infected-cell mRNA and was found to be identical to the in vivo antigen in antigenicity, in electrophoretic mobility, and in [35S]methionine-containing tryptic peptides. After labeling with [35S]methionine and several 3H-amino acids, this in vitro product gave an N-terminal sequence identical to that expected from one of the open reading frames (reading region 3) in the DNA sequence for region E4 of Hérissé et al. (Nucleic Acids Res. 9:4023-4042, 1981). It is likely that this antigen is the same as the nucleus-associated 11K polypeptide from E4 described by other authors.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

Combinatorics of peptide sextets encoded by a single microgene

Genetic information stored in DNA sequences is translated into protein by linking a triplet nucleotide sequence and an amino acid. Because the frames of the triplets can be configured in three ways, a total of six polypeptides, each with a different sequence, can be produced from a single double-stranded DNA molecule. We recently developed the MolCraft system [reviewed in K. Shiba, J. Mol. Catal. B 18 (2004) xxx], which enables us to make combinatorial polymers of three peptides translated from one strand of a double-stranded DNA molecule. To explore all the information that a single double-stranded DNA molecule encodes, we have now developed a new system, La-MolCraft, in which all six reading frames encoded by both strands are combinatorially polymerized using loop-mediated isothermal amplification of DNA (LAMP) [Nucl. Acids Res. 28 (2000) E63].     

Highly purified snRNPs retain antigenic determinants towards anti Sm antibodies

snRNPs purified by centrifugation in density gradients (BRUNEL et al. Nucl. Acids. Res., 1981, 9, 815–830 and SRI-WIDADA et al. Biochim. Biophys. Res. Commun., 1982, $\text{104}$, 457–462) retain an important part (46%) of their antigenicity towards anti-Sm antibodies indicating that antigenic determinants are among the small 4–5 polypeptides between 9 and 14,000 daltons found to be tightly associated with snRNAs. Furthermore, digestion of a great part of the RNA moiety does not abolish this antigenicity.As to the determinant for RNP antigen, our results suggest that it could depend on the three other proteins which are lost during the purification.     

Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.     

Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.     

Isolation and characterization of a partial cDNA clone for heparin cofactor II1

A human fetal liver cDNA library constructed in lambda gt11 was screened with affinity-purified rabbit antibodies raised against heparin cofactor II. One positive clone was plaque purified and the cDNA insert was completely sequenced. The clone encodes the C-terminal 167 amino acid residues of heparin cofactor II as well as the entire 3'-untranslated region of the message. Proline and leucine were identified in the P2 and P1 positions of the protease cleavage site, providing a possible explanation for the ability of heparin cofactor II to inhibit both thrombin and chymotrypsin-like proteases. The coding sequence is identical to that of the recently published human leuserpin 2 (Ragg (1986) Nucl. Acids Res. 14, 1073).     

Sequence organization of a viral DNA insertion present in the adenovirus-type-5-transfonned hamster line BHK268-C31

The hamster cell line BHK268-C31 contains two large viral inserts which both include sequences from the left-hand end of adenovirus type 5 (Ad5) DNA. One of these viral inserts has been cloned in the λ vector Charon 4A. Electron microscopic analysis and restriction enzyme mapping shows that the recombinant carries a 4.4-kb-long colinear segment of viral DNA, which is located between map positions 1.5 and 14.2 in the Ad5 genome. The junctions between viral DNA and flanking sequences have been sequenced and found not to show any specific features. One of the junctions is located in the E1 a coding region, 573 bp from the left-hand end of the Ad5 genome, whereas the other junction is situated in the coding region for polypeptide IVa2. The promoter region as well as the cap site for the mRNAs from region E la are thus missing from this insert and its role in viral transformation is unclear.