Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.     

Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence.

Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Electron microscopic demonstration of the presence of amplified sequences at the 5''-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome

The entire nucleotide sequence of the Ad. 2 EcoRI D fragment has been determined using the Maxam and Gilbert method. This sequence of 2678 bp contains informations relative to late mRNAs ending at position 78 and for which an AATAAA sequence corresponding to their 3' ends is found at residue number 833. Position of the PVIII mRNA is determined thus allowing deduction of the probable amino acid sequence of the PVIII protein. The position and the sequence of the first leader of early 3 mRNAs is determined as well as the sequence and position of the second early leader of region 3 mRNAs, which also correspond to the "y" leader of the fiber mRNA. Following the localization of an open reading frame in which an ATG could initiate protein synthesis it can be predicted that 3a, b, c mRNAs code for the 16K early protein and the probable amino acid sequence of this protein can be deduced. The CAGTTT sequence frequently present at the 5' end of a leader or of a mRNA body as well as the GGTGAG sequence which is found at the 3' end of several leaders were used to postulate the position of various early mRNAs of region 3 and to suggest the existence of an additional splicing event during the processing of mRNAs 3a, b and c. They were also used to predict the position of the additional "x" late leaders. The imbrication of information concerning (i) the family of late mRNAs ending at position 78, (ii) the position of the "x" leader and the "y" leader and (iii) the beginning of early region 3 is also depicted.     

Human interferon gamma is encoded by a single class of mRNA

Polyadenylated RNA from human peripheral blood lymphocytes and spleen cells, treated with different inducers for IFN-gamma production, was fractionated on denaturing sucrose gradients. Two IFN-gamma mRNA peaks at 12S and 16S were consistently observed. Nucleotide sequence analysis of cDNA clones showed that the 12S IFN-gamma mRNA from the different sources is identical to the gel fractionated 18S IFN-gamma mRNA which gave rise to the IFN-gamma cDNA clone p69 (1). Nucleotide sequence analysis of several IFN-gamma cDNA clones showed the presence of a CGA (Arg) codon at position 140 of mature IFN-gamma in contrast with the CAA (Gln) codon, which is found in p69 (1). Specifically primed cDNA extension on total induced polyadenylated RNA revealed the presence of a single mRNA species having a 5' untranslated region of 125-130 nucleotides. The nucleotide sequence of this region has been obtained. These data suggest that a single human IFN-gamma gene, which has very little polymorphism, gives rise to a single size class of mRNA.     

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.     

Leader sequences of eukaryotic mRNA can be simultaneously bound to initiating 80S ribosome and 40S ribosomal subunit

Binding of mRNA leader sequences to ribosomes was studied in conditions of a cell-free translation system based on wheat germ extract. Leader sequence of TMV mRNA (the so-called omega-RNA sequence) was able to bind simultaneously 80S ribosome and 40S ribosomal subunit. It was found that nucleotide substitutions in omega-RNA resulting in destabilization of RNA structure have no effect on the complex formation with both 80S ribosome and 40S ribosomal subunit. Leader sequence of globin mRNA is also able to form a similar joint complex. It is supposed that the ability of mRNA leader sequences to bind simultaneously 80S ribosome and 40S subunit is independent of leader nature and may reflect previously unknown eukaryotic mechanisms of translation initiation.     

Minus-strand copies of replicating coronavirus mRNAs contain antileaders.

The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.     

The sequence of the 3'' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.     

Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.