Biomedical subjects
Publications and source records attributed to S M Weissman.
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Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.
Cytoplasmic mRNA isolated from cells infected with SV40 was isolated by passage over oligo(dT)-cellulose columns. This RNA was annealed to SV40 DNA fragments produced by cleavage with EcoRII endonuclease. The RNA resistant to RNase digestion was analyzed by digestion with ribonucleases and oligonucleotide mapping. The results were compared with oligonucleotides from in vitro transcripts of the fragments and with whole genome SV40 cRNA which had been fractionated by hybridization to the fragments. The 5' ends of "early" and the large "late" SV40 mRNA, transcribed from opposite DNA strands, overlap for a region of 60 to 100 nucleotides. The region of overlap includes a portion of the segment of DNA containing the origin of DNA replication.
An earlier report (Subramanian, Dhar, and Weissman, 1977c) presented the nucleotide sequence of Eco RII-G fragment of SV40 DNA, which contains the origin of DNA replication. The nucleotide sequence of Eco RII-N fragment located next to Eco RII-G on the physical map of SV40 DNA is presented in this report. Eco RII-N is found to be a tandem duplication of the last 55 nucleotides of Eco RII-G. This tandem repeat is immediately preceded by two other reiterated sequences occurring within Eco RII-G, one of them being a tandem repeat of 21 nucleotides and the other a nontandem repeat of 10 nucleotides. These repetitive sequences occur in close proximity to the origin of DNA replication which is known to contain other specialized sequences such as a few palindromes (one of which is 27 long and possesses a perfect 2-fold axis of symmetry), one "true" palindrome, and a long A/T-rich cluster. The repeats (and the replication origin) occur within an untranslated region of SV40 DNA flanked by (the few) structural genes coding for the "late" proteins on the one side and that (those) coding for the "early" protein(s) on the other side. The reiterated sequences are comparable in some respects to repetitive sequences occurring in eucaryotic DNAs. Possible biological functions of the repeats are discussed.
The nucleotide sequence of 70 base pairs (bp) around 0.545 map units (Alu I C and B junction) of the genome from the single Eco RI cleavage site within SV40 DNA is presented. The mRNA transcribed from the early strand template from this stretch contains two copies of the nonsense triplet UAA in each of the three reading frames. Thus at least 25% of the early region of SV40 DNA does not code for the SV40 "A" protein, and the viral contribution to events in the lytic cycle and transformation may be more complex than is generally appreciated.
The primary structures of human and rabbit beta-globin mRNAs are compared. Using as a standard the extent of nucleotide substitutions inferred from the hypervariable amino acid residues of fibrinopeptides A and B, which are thought to change largely by neutral evolution, we show that not all silent mutations in globin mRNA are neutral. The divergence of the sequences is limited in part by the selective usage of synonymous codons. The divergent nucleotides tend to be distributed nonrandomly: in the coding region silent substitutions are most rare in segments that are also deficient in substitutions leading to replacements.
The nucleotide sequence of the region of DNA of simian virus 40 extending from 0.595 to 0.790 map unit has been derived. The sequence includes the DNA complementary to the 5' end of early mRNA and to the 5' end of some of the forms of late RNA. Because there are termination codons in all three phases in early and late RNA, there is a sequence of almost 800 nucleotides of simian virus 40 DNA that probably does not code for known viral proteins. The sequence spans the 5' end of the early mRNA at 0.67 map unit and overlaps a species of late RNA whose 5' end is at 0.65 map unit and whose 3' end is at 0.77 map unit. This RNA is retained on oligo(dT)-cellulose columns in high salt concentrations. Analysis of the sequence of late strand RNA suggests that this RNA is not covalently linked to the mRNA that encodes structural proteins. There is another species of late RNA of simian virus 40 whose 5' end is at 0.775 map unit. The nucleotide sequence of this region of simian virus 40 DNA contains several examples of repeated sequences, most of which are located in DNA that does not encode known peptides. These may be analogous to the reiterated sequences that have been described in animal cell DNA.
We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A).
We have determined the sequence of the portion of Simian Virus 40 (SV40) that codes for the major structural protein of the virus. The gene contains 361 codons. Synonym codons for an amino acid are not used randomly. The dinucleotide CG occurs only once and there is 2 to 1 preference for uridylic acid in the third position of codons.
Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.
The 16S late mRNA from SV40 directs the synthesis of the major viral structural protein, VP1. We have compared the oligonucleotides in the 16S mRNA with those that would be present in a transcript of the portion of SV40 DNA coding for VP1. The results indicate that a segment of about 200 nucleotides of RNA transcribed from a distant part of SV40 DNA have become linked to the transcript of VP1 codons by a bond resistant to phenol extraction and denaturation in formamide.
1. One-fifth to 1/4 of globin mRNA is untranslated sequence other than polyadenylic acid. 2. The untranslated sequences of mRNA vary markedly in their sequence and in their length. 3. Globin mRNAs demonstrate a marked bias in codon selection. 4. Viral mRNA shows a quite different pattern of codon selection; therefore, the selection of codons is not uniform for all mRNAs functioning in animal cells. 5. Elongated hemoglobin chains can be accounted for by frame-shift mutations, or point mutations within the normal termination codon. The additional amino acids are then coded for by sequences that are normally untranslated. 6. Certain hemoglobin deletion mutants occur at sites where there are partially reiterated sequences within the heomoglobin messenger RNA.
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The nucleotide of Bacillus stearothermophilus 5 S RNA is pC-C-U-A-G-U-G-A-C-A-A-U-A-G-C-G-(G-A-G-A-G-G-)-A-A-A-C-A-C-C-C-G-U-U-C-C-C-A-U-C-C-C-G-A-A-C-A-C-G-G-A-A-G-U-U-A-A-G-C-U-C-U-C-C-A-G-C-G-C-C-G-A-U-G-G-U-A-G-U-U-G-G-G-G-C-C-A-G-C-G-C-C-C-C-U-G-C-A-A-G-A-G-U-A-G-G-U-C-G-U-U-G-C-U-A-G-G-COH; the nucelotide sequence of Bacillus subtilis 5 S RNA is pU-U-U-G-G-U-G-G-C-G-A-U-A-G-C-G-A-A-G-A-G-G-U-C-A-C-A-C-C-C-G-U-U-C-C-C-A-U-A-C-C-G-A-A-C-A-C-G-G-A-A-G-U-U-A-A-G-C-U-C-U-U-C-A-G-C-G-C-C-G-A-U-G-G-U-A-G-U-C-G-G-G-G-G-U-U-U-C-C-C-C-C-U-G-U-G-A-G-A-G-U-A-G-G-A-C-G-C-C-G-C-C-A-A-G-COH. Comparison of the sequence of B. stearothermophilus 5 S RNA to the sequence of B. subtilis 5 S RNA and to that of Bacillus megaterium 5 S RNA (Pribula, C. D., Fox, G. E., Woese, C. R., Sogin, M., and Pace, N. (1974) FEBS Lett. 44, 322-323) indicates that the 5 S RNA isolated from the thermophile contains unique nucleotide sequences not found in 5 S RNAs isolated from the two mesophilic species of genus Bacillus.
Recent studies of the structure of messenger RNA have demonstrated the existence of untranslated sequences of the 3' and 5' end of the messages. In addition analysis of transcription in vitro has indicated that the nucleotide sequence U6 purine may be part of a transcription termination signal in prokaryotes. Recently it has been possible to determine the sequence of extensive portions of the DNA of SV40 virus. This article reviews the analogies between certain of these sequences and sequences available from prokaryotic messengers and DNAs. Unusual structures, including blocks of AT-rich and GC-rich segment sections and symmetric regions in the DNA near the origin of DNA replication, have been demonstrated and the distribution of stretches of 6 or more deoxyadenylic acids in the DNA of SV40 is consistent with some rho for these sequences in animal cells, either as terminators of transcription or as sites where degradation of transcripts is initiated or sites related to the selective rejection or degradation of transcipts.
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A major product of the transcription of bacteriophage lambda DNA in vitro is the 6S RNA. This article presents a detailed mapping of restriction endonuclease cleavage sites about the region of the 6S RNA template within the lambda genome. Restriction fragments defined by these sites have been used to localize the 6S RNA template within the physical and genetic maps of the lambda genome. Nucleotide sequence analysis of one of these fragments has largely confimed the nucleotide sequence of the 6S RNA reported previously and has indicated the sequence of DNA that immediately follows the 6S RNA template. This article reports the nucleotide sequence following a known site of transcription termination by RNA polymerase of Escherichia coli.