Paul Lebowitz

Heterogeneity and 5′-terminal structures of the late RNAs of simian virus 40

We have investigated the 5′-terminal structures of the late lytic RNAs of simian virus 40 (SV40) by binding the plus strand of small DNA fragments labeled in the 5′-terminal position with 32P to specific regions of cytoplasmic polyadenylated late RNA, extending these “primer” fragments in a 3′ direction with reverse transcriptase, fractionating the extended products on denaturing polyacrylamide gels and performing DNA sequence analyses on the extended products. Extension of a primer bound to the 5′ terminus of the body of 19 S RNA revealed a multiplicity of extended products. RNAs from which these products are derived fall into four classes, the first containing sequences colinear with SV40 DNA and the latter three containing splices which fuse the 5′ terminus of the 19 S body at residue 476 (0.765 map units) to residues 444, 291 and 212, respectively. Of 15 extended products we have analyzed, eight have 3′ termini with the sequence T-A(A). These termini lie at positions 243, 182, 110, 55, and 5189 on the SV40 genome. A number of lines of evidence, including nucleotide sequence analysis of late RNA labeled in vivo and the fact that the principal sequence in SV40 late mRNAs adjacent to capped structures is A-U(U), suggest that the termini at positions 243 and 182 correspond to the 5′ termini of discrete in vivo 19 S RNAs and that stops at the shorter positions may also correspond to the 5′ termini of specific in vivo species of 19 S RNA. An additional extended product was observed with a sequence colinear with SV40 DNA and a 3′ terminus of T-A-A at residue 548 (0.779 map units). It contains sequences complementary to the VP3 but not the VP2 initiation codons of 19 S RNA. For each of the three different gaps we have found in the late 19 S RNAs, as well as four additional gaps we have analyzed in the late 16 S and early 19 S RNAs of SV40, identical di-, tri-or tetranucleotide sequences lie on the ungapped precursor at sites which undergo splicing; these sequences may be involved in determining the specificity of the splicing reaction.

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

Abstract The reduced β-globin synthesis characterizing the β + thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β + thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β + -thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.

Hemophilus aegyptius restriction endonuclease cleavage map of the simian virus 40 genome and its colinear relation with the Hemophilus influenzae cleavage map of SV40

Abstract Cleavage of simian virus 40 DNA I with the restriction endonuclease from Hemophilus aegyptius yields 16 fragments of approximately 18 to 1520 nucleotides. These fragments have been ordered and precisely aligned with the fragments composing the H. influenzae and H. parainfluenzae endonuclease cleavage maps of SV40 † by a combination of two methods: (1) hybridization of radioactively labeled transcripts of Hae fragments to denatured Hin fragments; (2) reciprocal digestion of Hin fragments with H. aegyptius endonuclease and Hae fragments with the endonucleases from H. influenzae and H. parainfluenzae , followed by size analysis of the resultant products. In addition, nucleic acid hybridizations have been performed between the transcripts of certain Hae fragments and DNA from the adenovirus-SV40 hybrids, Ad2 + ND 1 , Ad2 + ND 4 and E46 + , which permit more precise localization of the distal termini of the SV40 segments within these hybrids on the map of the SV40 genome.

Transcription of simian virus 40 DNA by Escherichia coli RNA polymerase: Synthesis of a DNA-RNA hybrid and discrete RNAs under restrictive transcription conditions☆

Simian virus 40 DNA I has been transcribed with Escherichia coli RNA polymerase at substrate concentrations of 5.5 to 16.5 μm. Sucrose gradient and polyacrylamide gel analysis of the products of transcription reveals the presence of a 21 S template DNA-RNA hybrid molecule and a large number of discrete RNAs of 5 to 11 S. The hybrid molecule serves as an intermediate in transcription. The discrete RNAs are transcribed from all areas of the genome, are initiated at specific DNA loci, and probably arise largely as a result of enhanced specificity of termination.

COMPLEX STRUCTURES AND NEW SURPRISES IN SV40 mRNA11This work was supported by a grant from the American Cancer Society and by grant #CA-16038 from the National Cancer Institute, DHEW.

ABSTRACT We have determined the nucleotide sequences of many of the SV40 mRNAs produced in virus transformed cells, and early and late in lytically infected cells. Late lytic mRNAS exhibit a multiplicity of 5′ ends. 16S mRNAs all contain contiguous stretches of RNA coding for VP1 spliced onto three classes of 5′ terminal leaders. One class of leaders contains an intraleader splice while another contains a duplication of 93 nucleotides at the 3′ terminus of the leader. 19S mRNAs contain coding information for VP2 and VP3 and fall into four classes, one of which is comprised of unspliced species and three of which contain different 5′ terminal splices. 16 and 19S mRNAs share only one major leader. The late mRNA leaders are transcribed from a region of approximately 10% of the viral genome. SV40 large and small T antigen early mRNAs isolated from infected and transformed cells exhibit single well-defined overlapping splices and a limited degree of 5′ terminal heterogeneity.