Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA.

Fiber mRNA of adenovirus 2 has been used as a template for RNA-dependent DNA polymerase. The resulting cDNA/RNA hybrids have been inserted at the Pst I site of the plasmid vector pBR322 after A:T tailing. One recombinant plasmid, pJAW 43, has been characterized in detail and shown to contain sequences from the main body of fiber mRNA, the three leaders common to most late adenoviral mRNAs and a fourth leader found in some species of fiber mRNA. The complete DNA sequence of the leader region has been determined and does not contain the initiation codon AUG, although this codon does occur immediately downstream from the junction between the fourth leader and the main body of the fiber mRNA. The first leader (map coordinate 16.6) is 41 nucleotides long, the second (from 19.6) is 71 nucleotides, the third (from 26.6) is 88 nucleotides and the fourth (from 78.5) is 181 nucleotides. The location of junctions between viral leaders and intervening sequences has been determined by reference, where possible, to sequences of the adenovirus 2 genome. Although the presence of short repeated sequences at the boundaries of intervening sequences and leaders makes it impossible to locate the splice point unambiguously, all of the leader-intervening sequence junctions can be arranged to stress a common feature--the presence of the dinucleotides GT and AG at the 5' and 3' ends, respectively, of the intervening sequences. This prototype sequence, which has also been recognized at or near the splice points in other eucaryotic systems, is possibly part of a larger unit which serves as a recognition site for specific excision-ligation events that ultimately lead to the production of mature mRNAs.     

The genes of influenza virus

The 5′ terminal sequences of several adenovirus 2 (Ad2) mRNAs, isolated late in infection, are complementary to sequences within the Ad2 genome which are remote from the DNA from which the main coding sequence of each mRNA is transcribed. This has been observed by forming RNA displacement loops (R loops) between Ad2 DNA and unfractionated polysomal RNA from infected cells. The 5′ terminal sequences of mRNAs in R loops, variously located between positions 36 and 92, form complex secondary hybrids with single-stranded DNA from restriction endonuclease fragments containing sequences to the left of position 36 on the Ad2 genome. The structures visualized in the electron microscope show that short sequences coded at map positions 16.6, 19.6 and 26.6 on the R strand are joined to form a leader sequence of 150–200 nucleotides at the 5′ end of many late mRNAs. A late mRNA which maps to the left of position 16.6 shows a different pattern of second site hybridization. It contains sequences from 4.9?6.0 linked directly to those from 9.6?10.9. These findings imply a new mechanism for the biosynthesis of Ad2 mRNA in mammalian cells.     

All subgenomic mRNAs of equine arteritis virus contain a common leader sequence.

During the replication of equine arteritis virus (EAV) six subgenomic mRNAs are synthesized. We present evidence that the viral mRNAs form a 3'-coterminal nested set and contain a common leader sequence of 208 nucleotides which is encoded by the 5'-end of the genome. The leader is joined to the bodies of mRNA 5 and 6 at positions defined by the sequence 5' UCAAC 3'. The part of the leader sequence flanking the UCAAC motif is very similar to the 5'-splice site of the Tetrahymena pre-rRNA. A possible internal guide sequence has been identified 43 nucleotides downstream of the leader sequence on the genome. Hybridization analysis shows that all EAV intracellular RNAs contain the leader sequence. These data imply that the viral subgenomic mRNAs are composed of leader and body sequences which are non-contiguous on the genome.     

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5

We have determined the nucleotide sequence of a region of adenovirus type 5 (Ad5) DNA located between map positions 61.7 and 71.4, which covers the gene form the 72 kD DNA binding protein (DBP) and the sequence encoding the amino-terminal part of the 100 kD protein. Sequence analysis of cDNA copies of DBP mRNA revealed the existence of two abundant species of spliced mRNA molecules. One species consists of two short leader sequences from positions 75.2 (67 and 68 nucleotides long) and 68.8 (77 nucleotides long), respectively, and the main body of the RNA molecules. The other species contains only the leader sequence from position 75.2 and the main body. The amino acid sequence of DBP is encoded entirely by a long open reading frame of 1587 nucleotides in the main body of DBP mRNA. From the nucleotide sequence of the DBP gene it can be derived that DBP contains 529 amino acid residues and has an actual molecular weight of 59,049 daltons. The sites of mutation in the mutants H5hr404 and H5ts125 were determined at the nucleotide level. Single nucleotide alterations were detected in H5hr404 and H5ts125 in the sequences corresponding to the amino-terminal part and the carboxy-terminal part of DBP, respectively. The implications of these mutations are discussed.     

Two adenovirus mRNAs have a common 5′ terminal leader sequence encoded at least 10 kb upstream from their main coding regions

The messenger RNAs encoding two late adenovirus serotype 2 (Ad2) proteins, fiber and 100K, were purified by hybridization to restriction endonuclease fragments of Ad2 DNA followed by electrophoresis on polyacrylamide gels containing 98% formamide. The 5′ terminal oligonucleotides generated by RNAase T1 digestion of the messengers were selected by dihydroxyboryl-cellulose chromatography. Both mRNAs gave an identical 5′-undecanucleotide with the general structure 7mG^5′ppp^5′A^mC^(m)U(C₄,U₃)G. This undecanucleotide could be removed by mild RNAase treatment from the mRNA after hybridization to DNA fragments containing the main coding sequence of the messenger. In contrast, a small region defined by Bal I-E (14.7–21) protects this undecanucleotide from RNase. A second region contained within both Hind III-B (17–31.5) and Hpa I-F (25.5–27.9), although unable to protect the undecanucleotide, hybridizes to both fiber and 100K mRNAs and protects a similar sequence of 100–150 nucleotides. These observations suggest that both mRNAs contain a long common sequence, complementary to at least two different sites on the Ad2 genome remote from the start of these two genes. The implications of these findings are discussed, and a general mechanism is presented for the biosynthesis of mRNAs from larger precursor molecules, based on intramolecular ligation.     

Characterization of the simian virus 40-specific messenger RNAs isolated from HeLa cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4

Previous work has shown that cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses, Ad2⁺ND2 and Ad2⁺ND4 synthesize more than one SV40⁴ large T antigen-related protein. These proteins overlap in amino acid sequence and have their carboxy-terminal sequences in common (Mann et al., 1977). We have characterized the messenger RNAs coding for these SV40-specific proteins. By translating in vitro SV40-specific mRNA isolated from cells infected with these viruses we have shown that each SV40-specific protein can incorporate ³⁵S-labeled formyl methionine at its N-terminus donated by [³⁵S]-fmet-tRNA_f^met, demonstrating that each protein results from a de novo initiation event. Furthermore, analysis of the N-terminal tryptic peptides of these proteins indicates that each protein has a unique N-terminal peptide and therefore a unique initiation site for protein synthesis, with the possible exception of the 74,000 and 95,000 molecular weight proteins, which may have the same N-terminal sequence. Therefore, these proteins cannot be derived by proteolytic cleavage of a large precursor protein.The messenger activities for many of the hybrid virus proteins can be resolved by gel electrophoresis, demonstrating the presence of multiple SV40-specific mRNA species. This result is consistent with the possibility that each SV40-specific protein is coded by a distinct species of RNA.     

Conditional lethal mutants of adenovirus type 2-simian virus 40 hybrids. II. Ad2+ND1 host-range mutants that synthesize fragments of the Ad2+ND1 30K protein.

Adenovirus type 2 (Ad2) grows 1,000 times less well in monkey cells than in human cells. This defect can be overcome, not only upon co-infection of cells with simian virus 40 (SV40), but also when the relevant part of the SV40 genome is integrated into the adenovirus genome to form an adenovirus-SV40 hybrid virus. We have used the nondefective Ad2-SV40 hybrid virus Ad2+ND1, which contains an insertion of 17% of the SV40 genome, to isolate host-range mutants which are defective in growth on monkey cells although they grow normally on human cells. Like Ad2, these mutants are defective in the synthesis of late proteins in monkey cells. A 30,000-molecular-weight protein (30K), unique to Ad2+ND1-infected cells, can be synthesized in vitro, using Ad2+ND1 mRNA that contains SV40 sequences. 30K is not seen in cells infected with those host-range mutants that are most defective in growth on monkey cells, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of 30K. Genetic and biochemical analyses indicate that these mutants carry point mutations rather than deletions.     

Complete mitochondrial genome sequence of Catla catla and its phylogenetic consideration

Complete nucleotide sequence of mitochondrial genome (mitogenome) of the Catla catla (Ostariophysi: Cypriniformes: Cyprinidae) was determined in the present study. Its length is 16,594 bp and contains 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs and one non-coding control region. Most of the genes were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. The reading frames of two pair of genes overlapped: ATPase 8 with 6 and ND4L with ND4 by seven nucleotides each. The main non-coding region was 929 bp, with three conserved sequence blocks (CSB-I, CSB-II, and CSB-III) and an unusual simple sequence repeat, (TA)₇. Phylogenetic analyses based on complete mitochondrial genome sequences were in favor of the traditional taxonomy of family Cyprinidae. In conclusion present mitogenome of Catla catla adds more information to our understanding of diversity and evolution of mitogenome in fishes.     

Nucleotide sequence of Rous sarcoma virus RNA at the initiation site of DNA synthesis: The 102nd nucleotide is U.

The T₁ oligonucleotide in the genome Rous sarcoma virus (RSV) that corresponds to the initiation site of DNA synthesis in vitro was identified by hybridization of genome RNA with RSV strong stop DNA (the initial 101-nucleotide long fragment synthesized in endogenous reactions) and partially sequenced. The sequence of (C₂, U₂) A-U-U-U-G found corresponds to the d(A-A-T-G-A-A-G) sequence at the 5′ end of the DNA product plus the CA-OH sequence at the 3′ end of the tRNA^Trp primer. Therefore the nucleotide opposite the terminal A of the primer is the complementary U. Furthermore, no internal repetition of more than 30 nucleotides of the 5′ sequence could be detected.     

Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus

We have obtained the complete sequence of cloned full-length DNA (NS DNA) derived from influenza virus gene 8, which codes for two unique polypeptides, NS₁ and NS₂, and the sequence of the NS₂ mRNA. Previously we showed that the mRNA for NS₁ (～860 nucleotides) is colinear with the viral RNA and maps from 0.05?0.95 units of the cloned NS DNA, and the body of the NS₂ mRNA (～340 nucleotides) maps from 0.59?0.95 units, suggesting that the two mRNAs are 3′ co-terminal and share the same poly(A) addition site. Sequencing studies have shown that the NS₂ mRNA contains an interrupted sequence of 473 nucleotides. The nucleotide sequences at the junctions of the interrupted segment are similar to those of the consensus sequences at the splicing sites of intervening regions in eucaryotic mRNAs. The first ～56 virus-specific nucleotides at the 5′ end of the NS₂ mRNA are the same nucleotides as are found at the 5′ end of the NS₁ mRNA, and this leader sequence of the NS₂ mRNA contains the initiation codon for protein synthesis and coding information for nine amino acids which would be common to NS₁ and NS₂. In addition, both mRNAs contain 10–20 heterogeneous nonviral nucleotides at their 5′ ends. The ～340 nucleotide body region of the NS₂ mRNA can be translated in the +1 reading frame, and the sequence indicates that NS₁ and NS₂ overlap by 70 amino acids that are translated from different reading frames.     

The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: implications for the mechanism of DNA replication.

The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the adjacent HindIII-F fragment, extending from the right-hand terminus to the sequences from which the main body of the mRNA of early region 4 is transcribed. One of the origins of adenovirus DNA replication is located within this part of the genome. The sequencing results are discussed in relation to several models proposed for the mechanism of replication of linear DNA molecules, which invariably depend on the presence of specific arrangements of nucleotides at the termini of those linear DNAs.