Characterization of a major late herpes simplex virus type 1 mRNA

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.     

Herpes simplex virus type 1 HindIII fragment L encodes spliced and complementary mRNA species.

We have used DNA bound to cellulose to isolate and translate in vitro herpes simplex virus type 1 (HSV-1) mRNA's encoded by HindIII fragment L (mapping between 0.592 and 0.647), and 8.450-base-pair (8.45-kb) portion of the long unique region of the viral genome. Readily detectable, late mRNA's 2.7 and 1.9 kb in size encoding 69,000- and 58,000-dalton polypeptides, respectively, were isolated. A very minor late mRNA family composed of two colinear forms, one 2.6 kb and one 2.8 kb, was isolated and found to encode only an 85,000-dalton polypeptide. A major early mRNA, 1.8 kb in size encoding a 64,000-dalton polypeptide, was also isolated. High-resolution mapping of these mRNA's by using S1 nuclease and exonuclease VII digestion of hybrids between them and 5' and 3' end-labeled DNA fragments from the region indicated that the major early mRNA contained no detectable splices, and about half of its 3' end was complementary to the 3' region of the very minor 2.6- to 2.8-kb mRNA's encoded on the opposite strand. These mRNA's also contained no detectable splices. The major late 2.7-kb mRNA was found to be a family made up of members with no detectable splices and members with variable-length (100 to 300 bases) segments spliced out very near (ca. 50 to 100 bases) the 5' end.     

Organization of RNA transcripts from a vaccinia virus early gene cluster

The detailed organization of the RNAs transcribed from an early gene cluster encoded by vaccinia virus has been determined from the information derived from several complementary techniques. These include hybrid selection coupled with cell-free translation to locate DNA sequences complementary to mRNAs encoding specific polypeptides; RNA filter hybridization to size and locate on the DNA mature RNAs as well as higher-molecular-weight RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; and fractionation of hybrid-selected mRNAs in an agarose gel containing methyl mercury hydroxide followed by the cell-free translation of these mRNAs to definitively ascertain the size of the mRNA encoding each polypeptide. The early gene cluster is located between 21 and 26 kilobases from the left end of the vaccinia virus genome and is encoded by a 5.0-kilobase EcoRI fragment which spans the HindIII-N, -M, and -K fragments. Transcribed towards the left terminus are four mature mRNAs, 1,450, 950, 780, and 400 nucleotides in size, encoding polypeptides of 55, 30, 20, and 10 kilodaltons, respectively. These mRNAs are colinear with the DNA template and are closely spaced such that the 5' terminus of one mRNA is within 50 base pairs of the 3' terminus of the adjacent RNA. In addition to the mature size mRNAs, there are higher-molecular-weight RNAs, 5,000, 3,300, 2,350, 2,300, 1,800, 1,700, and 1,350 nucleotides in size. The 5' and 3' termini of the high-molecular-weight RNAs are coterminal with the 5' and 3' termini of the mature size mRNA. The implications of this arrangement and the biogenesis of these early mRNAs are discussed.