Altered utilization of splice sites and 5'' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.     

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.     

Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides

We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

Isolation of vesicular stomatitis virus defective interfering genomes with different amounts of 5''-terminal complementarity.

I isolated at least 30 different vesicular stomatitis virus defective interfering (DI) genomes, distinguished by chain length, by five independent undiluted passages of a repeatedly cloned virus plaque. Labeling of the 3' hydroxyl ends of these DI genomes and RNase digestion studies demonstrated that the ends of these DI genomes were terminally complementary to different extents (approximately 46 to 200 nucleotides). Mapping studies showed that the complementary ends of all of the DI genomes were derived from the 5' ends of the nondefective minus-strand genome. Regardless of the extent of terminal complementarity, all of the DI genomes synthesized the same 46-nucleotide minus-strand leader RNA.     

Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid

Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.