Genomic complexities of murine leukemia and sarcoma, reticuloendotheliosis, and visna viruses.

The genetic complexities of several ribodeoxyviruses were measured by quantitative analysis of unique RNase T1-resistant oligonucleotides from 60-70S viral RNAs. Moloney murine leukemia virus was found to have an RNA complexity of 3.5 x 10(6) daltons, whereas Moloney murine sarcoma virus had a significantly smaller genome size of 2.3 x 10(6). Reticuleondotheliosis and visna virus RNAs had complexities of 3.9 x 10(6), respectively. Analysis of RNase A-resistant oligonucleotides of Rous sarcoma virus RNA gave a complexity of 3.6 x 10(6), similar to that previously obtained with RNase T1-resistant oligonucleotides. Since each of these viruses was found to have a unique sequence genomic complexity near the molecular weight of a single 30-40S viral RNA subunit, it was concluded that ribodeoxyvirus genomes are at least largely polyploid.     

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.     

Molecular weight of DNA from actinophage MSP2.

Actinophage MSP2 is infectious for Streptomyces venezuelae S13. Based upon electron microscopy of coliphage T4 mixed with MSP2, MSP2 had a head about 48 +/- 2 nm wide and 87 +/- 5 nm long. DNA from polyoma virus and from coliphages T4 and T7 served as reference markers in estimating the molecular weight of MSP2 DNA from sedimentation in sucrose gradients. Denatured MSP2 DNA was estimated to be about 17 x 10(6) and double-stranded MSP2 DNA was about (36 +/- 1.6) x 10(6) in molecular weight.     

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.     

Molecular Weight of Two Oncornavirus Genomes: Derivation from Particle Molecular Weights and RNA Content

Sedimentation analysis and intensity fluctuation spectroscopy have been used in conjunction with the Svedberg equation to determine the particle molecular weights of Rous sarcoma virus (Prague strain) and avian myeloblastosis virus (BAI strain). The molecular weights of these two viruses are (294 +/- 20) x 10(6) and (256 +/- 18) x 10(6), respectively. Values for the molecular weight of the RNA contained in each particle have been calculated as (5.58 +/- 0.5) x 10(6) and (5.88 +/- 0.5) x 10(6). Since the proportion of the viral RNA represented by 4 to 7S low-molecular-weight material is known, the molecular weight of the 60 to 70S genomes may be calculated to lie in the range (3.8 +/- 0.3 to 4.8 +/- 0.4) x 10(6) for both particles. These estimates for the molecular weight of the 60 to 70S genome are much lower than previous estimates and fall within the range of current estimates of the size of a single 35S subunit. The implications of this finding are discussed in terms of current theories for the structure of the genome of RNA tumor viruses.     

Complexity and polyadenylic acid content of visna virus 60-70S RNA.

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.     

Genetic structure and transforming sequence of avian sarcoma virus UR2.

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 10(6), corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'- and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 12 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.     

Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70S RNA: Evidence for a Haploid Genome

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.     

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.     

Domain swapping in ribonuclease T1 allows the acquisition of double-stranded activity

A mutant of ribonuclease T1 (RNase T1), denoted RNase Talpha, that is designed to recognize double-stranded ribonucleic acid was created. RNase Talpha carries the structure of RNase T1 except for a part of its loop L3 domain, which has been swapped for a corresponding domain from alpha-sarcin. The RNase Talpha maintains the pleated beta-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase Talpha-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced K(m) value of 6.94 x 10(-7) M. When the stranded specificity is examined, RNase Talpha catalyzes the hydrolysis of guanine base not only of single-stranded but also, as by design, of double-stranded RNA. The change of stranded specificity suggests the feasibility of using domain swapping to make a substrate-specific ribonuclease. This study suggests that the loop L3 in RNase T1 can be used as a 'cassette player' for inserting a functional domain to make ribonuclease of various specificities.     

Methylation pattern of genomic RNA from Moloney murine leukemia virus.

32P- and methyl-3H-labeled 70S Moloney murine leukemia virus RNA was purified from virions produced in Moloney murine leukemia virus-infected mouse embryo cells. Primer-free RNA subunits obtained by heat treatment and zonal centrifugation were digested with RNase T2, and methylated oligonucleotides were chromatographed on DEAE-Sephadex in 7 M urea. Approximately one molecule of RNase T2-stable oligonucleotide (-5 charge) was isolated per subunit. Structural analysis indicated that the sequence of the oligonucleotide is m7GpppGmpCp. Analysis of the mononucleotide fraction isolated by DEAE-Sephadex chromatography of the RNase T2 digest identified 15 to 23 internal N6-methyladenylic acid molecules per subunit.     

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed.     

Size, Composition, and Structure of the Deoxyribonucleic Acid of Herpes Simplex Virus Subtypes 1 and 2

Studies of the size, composition, and structure of the deoxyribonucleic acid (DNA) of the F and G prototypes of herpes simplex virus (HSV) subtypes 1 and 2 (HSV-1 and HSV-2) showed the following. (i) As previously reported by Good-heart et al. HSV-1 and HSV-2 DNA have a buoyant density of 1.726 and 1.728 g/cm(3), corresponding to 67 and 69 guanine +/- cytosine moles per cent, respectively. The difference in guanine plus cytosine content of the DNA species was confirmed by the finding of a 1 C difference in T(m). (ii) The DNA from purified virus on cocentrifugation with T4 DNA in neutral sucrose density gradients sedimented at 55S, corresponding to 99 +/- 5 million daltons in molecular weight. HSV-1 and HSV-2 DNA could not be differentiated with respect to size. (iii) Cosedimentation of alkali-denatured DNA from purified virus with T4 DNA on alkaline sucrose density gradients consistently yielded several bands of single-stranded HSV DNA ranging from fragments 7 x 10(6) daltons to intact strands 48 x 10(6) daltons in molecular weight.     

Oligonucleotide mapping of non-radioactive virus and messenger RNAs.

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.     

Size and location of poly (A) in encephalomyocarditis virus RNA.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.     

More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA.

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.