Current approaches to analysis of the nucleotide sequence of DNA.
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Biomedical subjects
Publications and source records attributed to S M Weissman.
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Cloned human DNA fragments containing globin genes are transcribed in vitro to form discrete RNA species. One transcription unit is located approximately 1500 base pairs upstream from the G-gamma-globin gene. This transcript is partially homologous to a polymerase III template located approximately 1000 base pairs upstream from the delta-globin gene and to DNA located a short distance downstream from the beta-globin gene.
We have studied the structure of polyadenylated virus-specific cytoplasmic mRNA's in mouse and human cells transformed by simian virus 40 and in monkey cells infected with simian virus 40 in the presence of cytosine arabinoside by means of reverse transcriptase-catalyzed complementary DNA synthesis and complementary DNA sequencing. Abundant mRNA species containing splices from residues 4490 to 4557 (0.533 to 0.546 map units [m.u.]) and 4490 to 4837 (0.533 to 0.600 m.u.) were identified in both transformed and infected cells. Two principal reverse transcriptase stops were observed at the 5' termini of these mRNA's, both occurring with approximately equal frequency. The most distal of these stops was localized at residues 5152 to 5154 (0.660 m.u.), and the second was at residues 5147 to 5148 (0.659 m.u.). Several additional minor stops, between approximately 0.62 and 0.65 m.u., were also found on complementary DNA copied from transformed cell mRNA; in contrast, only one additional stop was present on complementary DNA copied from early lytic mRNA. These data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional 5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's. Transformed cell mRNA was also found to contain a single 3' terminus at positions 2504 and 2505 (0.153 m.u.); termini lying beyond this site were not detected.
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The 16 S mRNA that directs the synthesis of VP1, the major structural protein of Simian virus 40 (SV40), is composed of a leader sequence of 203 nucleotides joined to a transcript that contains all the codons for VP1. The leader is transcribed from DNA between 0.723 and 0.762 map units while the VP1 coding transcript is copied from DNA from 0.94 to 0.17 map units. The leader sequence joins the transcript of the coding region at a position 42 nucleotides upstream from the initiator codon for VP1 by a 3':5'-phosphodiester linkage. Longer leader sequences also occur in a portion of SV40 late mRNA.
The nucleotide sequence of SV40 DNA was determined, and the sequence was correlated with known genes of the virus and with the structure of viral messenger RNA's. There is a limited overlap of the coding regions for structural proteins and a complex pattern of leader sequences at the 5' end of late messenger RNA. The sequence of the early region is consistent with recent proposals that the large early polypeptide of SV40 is encoded in noncontinguous segments of DNA.
The 3' ends of the mRNA coding for the early and late proteins of SV40 DNA overlap. We have analyzed the restriction endonuclease fragment of SV40 DNA complementary to the 3' untranslated ends of the mRNA and the codons for the COOH-terminal amino acids of early and late protein. The sequence of this DNA fragment is presented.
The nucleotide sequence for two-thirds of restriction endonuclease fragment EcoRII-F and part of RII-H of SV40 DNA is presented. This segment of SV40 DNA is complementary to the sequence near the 3' end of early mRNA. This sequence could be translated in one reading frame to form a large protein. However, in a second translational frame there are four AUG codons followed by 91 sense triplets, followed by a termination codon. These results provide the sequence for the entire 3' untranslated ends of SV40 early and late mRNAs and for the DNA beyond the 3' ends of the mRNAs. The ends of early and late mRNA are transcribed from the opposite strands of the same segment of DNA. At or beyond the 3' ends of both early and late mRNA are sequences whose transcripts would include uridylic acid-rich products.
We have used a combination of techniques of DNA and RNA sequence analysis to determine the nucleotide sequence of the portion of simian virus 40 DNA preceding and encoding the 5' end of mRNA for the structural protein VP2 of simian virus 40. Comparison of the sequence with those found in polyadenylated RNA in the cytoplasm of infected cells RNA shows that the transcript of sequences preceding the structural gene is more abundant than the transcript containing the codons for the protein. Between the abundant transcript of sequences preceding the coding region and the less abundant transcript of the coding region there is a short sequence whose transcript is not detected.
We have determined the nucleotide sequence of the DNA of simian virus 40. The proceeding report (Dhar, R., Reddy, V.B., and Weissman, S.M. (1978) J. Biol. Chem. 253, 612-620) presents the sequence of a portion of the simian virus 40 DNA that overlaps the region encoding the 5' end of the minor structural protein VP2. We report here the sequence of the remainder of the genes for minor structural proteins VP2 and VP3. The results indicate that the mRNA for the two proteins is read in the same phase and the initiation site for VP3 lies within the structural gene of VP2. The codons of the COOH-terminal amino acids of VP2 and VP3 are read in a second phase as the codons of the NH2-terminal amino acids of VP1.
In order to investigate the 5' terminal structural heterogeneity of the 16S size class of SV40 late RNA, we have bound an SV40 DNA fragment labeled at its 5' termini with P32 to the .939-.945 map unit region of late lytic cytoplasmic polyadenylated RNA, used reverse transcriptase to prepare cDNA copies of the 5' termini of this RNA, separated the cDNA products on an 8% polyacrylamide-7 M urea gel and subjected these products to nucleic acid sequence analysis. A number of discrete cDNAs were obtained. Analysis of these cDNAs has suggested the presence of three categories of 16S species all containing the same body extending from residues 1381-2592 (.939-.170 m.u.) but differring in the structure of their leader segments. Members of the first category contain leaders which are colinear with SV40 DNA, have a common 3' terminus at residue 444 and extend varying distances in a 5' direction. The most abundant 16S species contains a leader of 203 nucleotides and is a member of this group. RNAs of the second category contain leaders with an internal gap between residues 211-352. The single RNA comprising the third category contains a leader with a tandem repetition of nucleotides 351-443 at the 3' terminus of its leader.
Double stranded human globin cDNA was synthesized by use of viral reverse transcriptase from globin mRNA of cord blood of premature infants requiring exchange transfusions. The cDNA was introduced into plasmids and the recombinant DNA plasmids used to transform E. coli X1776. A number of transformants were obtained. Plasmid DNA from selected colonies was isolated and characterized for the type of globin cDNA it contained by three types of procedures: 1) hybridization to previously characterized 3H-labeled alpha,beta and gamma cDNA; 2) analysis of the size and nature of fragments produced by digestion of the plasma DNA by different restriction endonucleases; and 3) by rapid DNA sequence analysis of selected DNA fragments produced by restriction endonuclease digestion. Analysis by these techniques of plasmid DNA from different colonies has definitively identified the presence of human alpha, beta or gamma cDNA sequences in different plasmids.
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VA-RNA I is one of the very few RNA species produced in animal cells whose transcriptional initiation site is known precisely. We have analyzed the nucleotide sequence of the DNA preceding the 5' end of VA-RNA I and compared it with known prokaryotic promoters and presumptive eukaryotic promoters.
VA-RNA I is a low molecular weight RNA produced in large amounts in cells infected with adenoviruses. The 3' terminus of this RNA may represent a transcription termination site. We have demonstrated that this RNA occurs in infected cells in several forms which differ in the number of uridylic acid residues at the 3' ends. The nucleotide sequence of a DNA fragment overlapping the 3' end of VA-RNA I has been determined. The DNA could encode up to 4 uridylic acid residues at the 3' end of the RNA. The DNA sequence shows some similarity to known transcription termination sequences in prokaryotic systems.
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Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.