Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Structural Characteristics and Nucleotide Sequence Analysis of Genomic RNA from RD-114 Virus and Feline RNA Tumor Viruses

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.     

Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to (3)H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain "defective" strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.     

Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

Separation of Reticuloendotheliosis Virus from Avian Tumor Viruses

Velocity sedimentation and isopycnic density gradient centrifugation indicate that reticuloendotheliosis virus has a different mass and buoyant density than members of the avian tumor virus group. The group-specific antigen of the avian tumor virus group was not detected in concentrated and purified reticuloendotheliosis virus preparations.     

Suppression of a Morphogenic Mutant in Rous Sarcoma Virus Capsid Protein by a Second-Site Mutation: a Cryoelectron Tomography Study

Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing ∼30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).In the first step of retrovirus maturation, the Gag precursor polyprotein is processed into three main components: the matrix (MA), capsid (CA), and nucleocapsid (NC). Of these, ∼1,000 to 2,000 copies of CA assemble into a capsid shell, enclosing the dimeric RNA genome and viral replication enzymes, while leaving a sizable population of unassembled CA subunits (5, 24). A properly formed capsid is thought to be essential for infectivity. The CA proteins of different retroviruses are quite uniform in size, ∼230 to 240 amino acids, but exhibit little sequence similarity except in the major homology region (MHR). Nevertheless, they share a structure with two domains (the N-terminal and C-terminal domains [NTDs and CTDs, respectively]) of conserved folds, connected by a short linker (2, 3, 8, 12, 17, 21, 22, 28). The NTDs form hexameric and pentameric rings that associate via homodimeric interactions between CTDs present in neighboring rings. Recently, structural evidence for a third interaction between the NTDs and the CTDs has been presented (10, 16, 31).The observed conservation of the MHR sequence points to its functional importance; in particular, the MHR is thought to play key roles, both in Gag assembly to produce immature virions and subsequently in the assembly of capsids within maturing virions. Consistent with this, several studies have reported adverse effects of point mutations in the MHR: certain mutations block the assembly of Gag proteins, whereas others have no evident effect on Gag assembly, genome incorporation, or budding but yield virus-like particles that are noninfectious or poorly infectious (1, 7, 13, 26, 30, 35-37). Starting with mutations of the latter kind, a series of second-site suppressors have been isolated that partially restore infectivity. Of these, two mapped in the NTD (A38V and P65Q), three in the dimerization helix in the CTD (F193L, R185W, and I190V), and one in the cleavage site between CA and the downstream peptide (S241L) (4, 7, 13, 25). The rescue of the MHR mutants by suppressing mutations was found not to be allele specific (25). The lack of allele specificity and the temperature sensitivity of some MHR mutations and their suppressors suggest that the affected residues are important for achieving a conformation(s) needed for proper assembly.In a previous study, we used cryoelectron tomography (cryo-ET) to characterize the pleiomorphic variability of wild-type Rous sarcoma virions (6, 20). Some 80% of them were found to contain capsids, which could be tubular, irregular polyhedra, or “continuous curvature” capsids, lacking angular vertices. The capsid type was found to correlate with the number of glycoprotein spikes per virion and with the efficiency of assembly, or conversely, the size of the pool of unassembled CA subunits contained within a virion. Based on these observations, we posited that in capsid assembly in situ, which is necessarily a nucleated process, different kinds of nucleation complexes initiate assembly of the various kinds of capsids. We also observed that tubular and some continuous curvature capsids had little internal material, suggesting that packaging of the viral ribonucleoprotein (RNP) had failed. Accordingly, and to accommodate the extensive polymorphism that was observed, we posited that a viable core is one with a closed capsid (of any morphology) that has successfully packaged its RNP. Subsequent in vitro studies have supported and clarified the perception of CA assembly as a variably nucleated process. Specifically, (i) it has been demonstrated that CA protein can assemble in a nucleation-driven manner into a variety of capsid-related structures (32, 33), and (ii) the mutation F167Y has been found to hamper nucleation of CA assembly in vitro, while the A38V suppressor strongly promotes assembly. Thus, CA-A38V assembles exceedingly rapidly, and in the double mutant, the A38V change overcomes the nucleation defect caused by F167Y.We have now further investigated the relationships among capsid morphology, nucleated assembly, and infectivity by performing a cryo-ET analysis of virions produced by the temperature-sensitive double mutant in which F167Y is complemented with the suppressor A38V; at the permissive temperature, the infectivity of the double mutant is restored to about 70% of wild type (25). Prior observations (32, 33) allowed the prediction that capsid assembly in vivo would be impaired in initiation for F167Y and for F167Y-A38V (abbreviated hereafter as FY/AV) at the nonpermissive temperature. Expectations for the double mutant at the permissive temperature were less clear: capsids should be produced, but this process might be altered in some way, as infectivity is lower than in the wild type. Because infectivity as measured could also be affected by Gag-related functions occurring earlier in the replication pathway, i.e., in viral budding or in proteolytic processing, we also compared the rates of virus growth, terminal Gag cleavage, and budding efficiency under these conditions for both the mutants and the wild-type virus.     

Comparison of Rous Sarcoma Virus-Specific Deoxyribonucleic Acid Polymerases in Virions of Rous Sarcoma Virus and in Rous Sarcoma Virus-Infected Chicken Cells

Labeled virions of Rous sarcoma virus (RSV) were disrupted with detergent and analyzed on equilibrium sucrose density gradients. A core fraction at a density of approximately 1.24 g/cc contained all of the (3)H-uridine label and about 30% of the (3)H-leucine label from the virions. Endogenous viral deoxyribonucleic acid (DNA) polymerase activity was only found in the same location. Additional ribonucleic acid (RNA)- and DNA-dependent DNA polymerase activities were found at the top of the gradients. RNA-dependent and DNA-dependent DNA polymerase activities were also found in RSV-converted chicken cells. Particles containing these activities were released from cells by detergent and were shown to contain viral RNA. These particles were analyzed on equilibrium sucrose density gradients and were found to have densities different from virion cores.     

Properties and Location of Poly(A) in Rous Sarcoma Virus RNA

The poly(A) sequence of 30 to 40S Rous sarcoma virus RNA, prepared by digestion of the RNA with RNase T(1), showed a rather homogenous electrophoretic distribution in formamide-polyacrylamide gels. Its size was estimated to be about 200 AMP residues. The poly(A) appears to be located at or near the 3' end of the 30 to 40S RNA because: (i) it contained one adenosine per 180 AMP residues, and because (ii) incubation of 30 to 40S RNA with bacterial RNase H in the presence of poly(dT) removed its poly(A) without significantly affecting its hydrodynamic or electrophoretic properties in denaturing solvents. The viral 60 to 70S RNA complex was found to consist of 30 to 40S subunits both with (65%) and without (approximately 30%) poly(A). The heteropolymeric sequences of these two species of 30 to 40S subunits have the same RNase T(1)-resistant oligonucleotide composition. Some, perhaps all, RNase T(1)-resistant oligonucleotides of 30 to 40S Rous sarcoma virus RNA appear to have a unique location relative to the poly(A) sequence, because the complexity of poly(A)-tagged fragments of 30 to 40S RNA decreased with decreasing size of the fragment. Two RNase T(1)-resistant oligonucleotides which distinguish sarcoma virus Prague B RNA from that of a transformation-defective deletion mutant of the same virus appear to be associated with an 11S poly(A)-tagged fragment of Prague B RNA. Thus RNA sequences concerned with cell transformation seem to be located within 5 to 10% of the 3' terminus of Prague B RNA.     

Virion-Associated RNA Primer for Rous Sarcoma Virus DNA Synthesis: Isolation from Uninfected Cells

Uninfected chicken, duck, rat, and human fibroblast cells in culture contained a tRNA-like RNA molecule which was structurally identical to a virion-associated RNA primer for in vitro Rous sarcoma virus DNA synthesis. This primer RNA appeared to be a normal tRNA of these cells. It was not found in a number of lower eukaryotic cells or in Escherichia coli.     

Lack of Sequence Homology Between the 70S RNA of Various RNA Tumor Viruses and the DNA of Simian Virus 40 or Polyoma Virus

No significant hybridization was detected of DNA from simian virus 40 or polyoma virus, and of 70S RNA from avian myeloblastosis virus, murine leukemia virus (Rauscher), murine sarcoma virus (Kirsten), RD-114B, simian sarcoma virus-1, or Mason-Pfizer virus.     

Characterization of the Low-Molecular-Weight RNAs Associated with the 70S RNA of Rous Sarcoma Virus

Two low-molecular-weight RNAs are associated with the 70S RNA complex of Rous sarcoma virus: a previously described 4S RNA and a newly identified 5S RNA. The 4S RNA constitutes 3 to 4% of the 70S RNA complex or the equivalent of 12 to 20 molecules per 70S RNA. It exhibits a number of structural properties characteristic of transfer RNA as revealed by two-dimensional electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the 4S RNA species. The 5S RNA is approximately 120 nucleotides in length, constitutes 1% of the 70S RNA complex or the equivalent of 3 to 4 molecules per molecules of 70S RNA, and is identical in nucleotide composition and structure to 5S RNA from uninfected chicken embryo fibroblasts. Melting studies indicate that the 5S RNA is released from the 70S RNA complex at the same temperature required to dissociate 70S RNA into its constituent 35S subunits. In contrast, greater than 80% of the 4S RNA is released from 70S RNA prior to its conversion into subunits. The possible biological significance of these 70S-associated RNAs is discussed.     

Relationship of Reticuloendotheliosis Virus to the Avian Tumor Viruses: Nucleic Acid and Polypeptide Composition

The RNA content and polypeptide composition of reticuloendotheliosis virus (REV) was compared to that of C-type RNA tumor viruses. Two RNA species with approximate sedimentation values of 64S and 4S were observed after sucrose gradient centrifugation of RNA extracted from purified REV. The high-molecular-weight RNA species of REV sedimented slightly faster than that of the Bryan strain of Rous sarcoma virus (RSV). Although these characteristics were consistent with those of other C-type RNA tumor viruses, significant differences were observed when the polypeptide composition of REV was compared with that of RSV possessing envelope determinants of Rous-associated virus RAV-2 and RAV-3. Five polypeptides of which two were glycosylated were resolved by polyacrylamide gel electrophoresis. The major nonglycosylated polypeptide of REV did not comigrate with that of RSV (RAV-2)-RSV(RAV-3). The majority of the group-specific antigen reactivity resides in this major nonglycosylated polypeptide of avian tumor viruses and comigrates when proteins of several avian tumor viruses are subjected to coelectrophoresis. This difference in the migration of the major polypeptide of REV and RSV(RAV-2)-RSV(RAV-3) may explain the absence of avian tumor virus group-specific antigen in REV.     

Detection of Avian Tumor Virus RNA in Uninfected Chicken Embryo Cells

Uninfected chicken embryo cells were analyzed for the presence of viral ribonucleic acid (RNA) by molecular hybridization with the single-stranded deoxyribonucleic acid (DNA) product of the RNA-dependent DNA polymerase contained in avian sarcoma-leukosis virions. Viral RNA was detected in all cells which contained the avian tumor virus group-specific antigen and the virus-related helper factor. The amounts of viral RNA in these cells ranged from approximately 3 to 40 copies of viral-specific sequences per cell. In general, the viral RNA content correlated with the level of helper activity in the cells. Cells infected with Rous-associated virus 2 contained 3,000 to 4,000 copies of viral RNA per cell. RNA from these infected cells hybridized with nearly 100% of the viral (3)H-DNA. By contrast, a maximum of less than 50% hybridization was obtained with RNA from the uninfected helper-positive cells, suggesting that not all of the viral RNA sequences were present in these cells. No viral RNA was detected in cells which lacked group-specific antigen and helper activity. Under the conditions used in these studies, less than 0.3 viral genome equivalents of RNA per cell would have been detected.     

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5′end of the viral gRNA, known as the packaging signal (ψ), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to ψ, as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Rev-responsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16, 35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17, 18, 37, 41). Simple retroviruses do not encode Rev-like regulatory proteins, so other strategies for the export of unspliced viral RNAs are needed. For Mason-Pfizer monkey virus, a cis-acting constitutive transport element induces nuclear export of the unspliced viral RNA in a process mediated by the cellular mRNA nuclear export factor TAP (5, 25, 46, 63). In RSV, an RNA element composed of either of the two direct repeats flanking the src gene mediates the cytoplasmic accumulation of unspliced viral RNA by using host export proteins TAP and Dpb5 (29, 42, 44).The findings of recent studies suggest that specific RNA export pathways direct viral gRNA to sites of virion assembly (56); for example, HIV-1 gRNA export out of the nucleus by the Rev-RRE-CRM1 complex is required for the proper subcellular localization of Gag and efficient virus particle production (26, 57). In the case of RSV, little is known about the trafficking of the viral RNA destined for virion encapsidation or the effects of the gRNA nuclear export pathway on Gag trafficking and virus particle production. However, we do know that RSV Gag enters the nucleus during infection, owing to nuclear localization signals (NLSs) in the matrix (MA) and NC domains. The nuclear localization of Gag is transient, and export is mediated by a CRM1-dependent NES in the p10 region (6, 52, 53). Thus, it is feasible that Gag may facilitate the nuclear export of the gRNA, either directly or indirectly, to promote particle assembly (53).In support of this idea, Gag mutants engineered to be more efficiently directed to the plasma membrane than wild-type Gag by the addition of the Src membrane-binding domain (in Myr1E virus) or by the insertion of extra basic residues (in SuperM virus) are not concentrated in nuclei when cells are treated with the CRM1 inhibitor leptomycin B (LMB) (8, 20, 53). Moreover, Myr1E and SuperM virus particles incorporate reduced levels of viral gRNA compared to the levels incorporated by wild-type particles. Thus, there is a correlation between the nuclear transit of Gag and gRNA packaging, although the Myr1E and SuperM viruses may be deficient in gRNA encapsidation because they are transported to the plasma membrane too rapidly (8). To test the hypothesis that the loss of Gag nuclear trafficking is responsible for the gRNA packaging defect, we used a gain-of-function genetic approach whereby a heterologous NLS was inserted into Myr1E Gag, yielding mutant virus Myr1E.NLS. Our results revealed that restoring the nuclear trafficking of Myr1E Gag also restored the incorporation of gRNA into mutant virus particles.     

Transcription of DNA from the 70S RNA of Rous Sarcoma Virus II. Structure of a 4S RNA Primer

The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways-by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5' terminus and CpCpA(OH) at the 3' terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (Psip, Psip, Cp) Gp (possibly in place of the characteristic sequence GprTpPsipCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro.