Faithful Transcription Research Articles

The Poetry Notebooks of Ralph Waldo Emerson

Published here in full are Ralph Waldo Emerson's nine poetry notebooks, the single greatest source of information about his creative habits in poetry. Emerson kept rough drafts, revised versions, and fair copies of hundreds of poems in these notebooks, so that the genesis and development of poems both famous and obscure can be traced closely. notebooks have been remarkably little consulted, primarily because their unedited textual condition makes them difficult to use. This edition makes them accessible to scholars by presenting a faithful transcription of each notebook, a detailed analysis of the history of each poem, an introduction, and a cross-referenced index.For this edition, the editors have followed the high standards of textual practice developed for Harvard University Press's edition of The Journals and Miscellaneous Notebooks of Ralph Waldo Emerson. That editorial approach makes possible a logical, clear presentation of material that Emerson often jotted down in segments or with multiple erasures and insertions.Because it allow scholars to examine as never before the many facets of Emerson the poet, The Poetry Notebooks of Ralph Waldo Emerson will be a major impetus to study of the man considered by many to be America's greates thinker.

Activation of cryptic promoters of human c-myc genes in microinjected Xenopus laevis oocytes

The human cellular oncogene c-myc contains two promoters at the 5′ end of its first non-coding exon. Cryptic promoters located within the first intron are also activated to synthesize aberrant c-myc mRNAs in some Burkitt lymphomas with a t(8: 14) chromosome translocation in which a part of the gene structure, often the 5′ non-coding exon, is truncated. We have shown elsewhere that microinjected plasmid DNA carrying a normal, intact human c-myc gene directs efficient faithful transcription from its own two promoters in Xenopus laevis oocytes. Here, I have investigated the expression of different recombinants carrying various constructs of the c-myc, gene in frog oocytes in order to understand the activation mechanism of those cryptic promoters. Aberrant c-myc transcripts initiating from cryptic promoters within the first intron are undetectable when the intact or truncated c-myc gene construct is used. However, the cryptic promoters can be activated in Xenopus oocytes if the truncated c-myc gene construct is fused with simian virus 40 sequences containing a 21 base-pair repeat and the replication origin. Xenopus oocytes will be useful for further investigation of enhancing elements involved in the translocated and activated c-myc genes in Burkitt lymphoma cells.

Analysing Conversation in Fiction: The Christmas Dinner Scene in Joyce's: "Portrait of the Artist as a Young Man"

In this article I propose to demonstrate some of the ways in which the theories and principles developed by linguists and conversational analysts for the systematic study of discourse and natural conversation may be illuminatingly applied to the stylistic and structural study of a fictional conversation within a literary text. I acknowledge at the outset that natural and fictional conversation differ in many ways. It is not merely that in fiction the talk is tidied-up, that there are relatively few unclear utterances, overlaps, false-starts, hesitations, and repetitions: there are also literary conventions at work governing the fictional representation of talk, so that the rendered text is quite different from a faithful transcription of a natural conversation. However certain structural and functional

34] Vaccinia virus as an expression vector

The chapter describes vaccinia virus as an expression vector. It describes the methodology for using vaccinia virus to express foreign genes. These recombinant vaccinia viruses can be used for several purposes:(1)understanding the genetic regulation involved in the expression of endogenous vaccinia functions, (2) determining the fate of foreign gene products in a background free of native influences, (3) producing biologically active molecules, (4) elucidating the roles of pertinent antigens in eliciting defined immunological responses, (5) producing live recombinant vaccines, and (6) potentially using gene replacement therapy. The chapter discusses the biology of poxviruses stating that they comprise a large group of complex animal viruses, whose genetic information is contained within a double-standed DNA molecule and whose replication is confined to the cytoplasm of the infected cell. The chapter describes the principle of the method regarding the general protocol for the insertion of foreign genes into vaccinia virus. Vaccinia displays a number of characteristics, which make it an ideal expression vector. Some of these are as follows:(1) the ability to incorporate large amounts of exogenous DNA, (2) the faithful transcription of the exogenous genes such that the RNAs are translated into protein, which resemble the native product in structure, function, and localization, (3) the cytoplasmic site of vaccinia replication, allowing gene expression to proceed without interference by the host genome, and (4) the potentiality as a live recombinant vaccine as a target against human and veterinary pathogens. The future research efforts will focus on the manipulation of the vaccinia genome with respect to defining the genetic parameters, which control the expression of both endogenous and exogenous genes.

Injection of antibodies into the nucleus of amphibian oocytes : an experimental means of interfering with gene expression in the living cell

ABSTRACT The chromatin constituents associated with transcriptionally active genes can be studied by a variety of experimental approaches. Most of the methods used involve the isolation of active chromatin fractions followed by biochemical analysis, or are based on in vitro assays with the ultimate goal of reconstituting faithful transcription by adding purified and defined components to the transcriptional machinery (for reviews see Mathis, Oudet & Chambon, 1980; Rose, Stetler & Jacob, 1983a; Beroldingen et al. 1984; Reeves, 1984; Dynan & Tjian, 1985; Sentenac, 1985). Since these approaches require isolation of chromatin or of individual components therefrom, possible structural rearrangements or selective losses of certain constituents cannot a priori be excluded. Therefore it often remains questionable whether the results obtained in vitro reflect the actual situation in the living cell.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

Analysis of RNA polymerase III transcription complexes by gel filtration.

We studied the in vitro assembly and stability of RNA polymerase III transcription complexes on the 5 S RNA and tRNAMet genes of Xenopus and the VA genes of adenovirus. Complete and partial assemblies were formed on these genes using transcription factor IIIA from Xenopus ovaries and factors IIIB and IIIC from HeLa cells. The complexes were purified away from unbound factors by filtration through Sepharose 4B columns and then assayed for transcription in the presence of Xenopus polymerase III. The 5 S gene complexes were also investigated using a postlabeling DNase I footprinting technique that we devised. The binding of factor IIIA to the 5 S gene facilitated the binding of factor IIIC; this subassembly was required for factor IIIB to bind. On the VA I and tRNA genes, factor IIIC alone bound and allowed IIIB to bind. RNA polymerase bound last to form a preinitiation complex, but it was less stably affixed than any of the factors. The complete factor complexes on the 5 S and VA I genes were strikingly stable to brief exposure to high salt concentrations, and the stability of the factor IIIB interaction was limiting. Two modes of IIIC binding were distinguished that differed in stability and specificity. Assembly of the complexes did not require ATP, and faithful transcription occurred when adenyl-5'-yl imidodiphosphate was substituted for ATP.

A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro.

Faithful transcription of human mitochondrial DNA has been reproduced in vitro, using a fraction of mitochondrial proteins capable of accurate initiation at both the heavy- and light-strand promoters. Here we report the initial dissection of this system into two nonfunctional components which, upon mixing, reconstitute promoter-specific transcriptional capacity in vitro. One of these components copurifies with the major nonspecific RNA polymerase activity, suggesting its identity. The other component lacks significant polymerase activity, but contains a protein or proteins required for accurate initiation at the two individual promoters by isolated mitochondrial RNA polymerase. This factor facilitates specific transcription, but has little or no effect on nonspecific transcription of a synthetic copolymer (poly(dA-dT)), indicating a positive role in proper promoter recognition. The transcription factor markedly stimulates light-strand transcription, but only moderately enhances transcription initiation at the heavy-strand promoter, suggesting different or additional factor requirements for heavy-strand transcription. These requirements may reflect the functional differences between heavy- and light-strand transcription in vivo and, in particular, the role of the light-strand promoter in priming of heavy-strand DNA replication.

Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.

Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.

In vitro transcription of a cloned vaccinia virus gene by a soluble extract prepared from vaccinia virus-infected HeLa cells.

Faithful transcription of a vaccinia virus gene was accomplished in vitro by using a soluble extract prepared from vaccinia virus-infected HeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by using template DNA restricted within the transcribed region. The vaccinia virus gene was not transcribed by extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA polymerase, nor was a clone of adenovirus 2 DNA bearing the major late promoter transcribed by the extract from vaccinia virus-infected HeLa cells. Thus, infection by vaccinia virus altered cellular transcriptional specificity to favor expression of vaccinia virus genes. RNA synthesis by the infected cell extract was resistant to alpha-amanitin but strongly inhibited by beta, gamma-imido ATP and novobiocin.

Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences.

Faithful initiation of ribosomal RNA transcription from cloned DNA by purified RNA polymerase I.

A faithful transcription system for ribosomal RNA genes has been developed by using components from the small free-living amoeba Acanthamoeba castellanii. The system utilizes protein-free recombinant DNA as a template and in addition requires a crude cell-free extract containing RNA polymerase I and a transcription initiation factor (TIF-I). The transcript is initiated at the same position as the in vivo precursor ribosomal RNA: templates truncated at various sites downstream of the transcription start site give rise to only the predicted runoff RNA transcripts, and the runoff transcript produced has a 5'-terminus identical with the 5'-terminus of the isolated ribosomal RNA precursor. Faithful initiation can be elicited by the DNA sequence extending from -55 to +19 in the template. Subclones containing this sequence yield only the predicted runoff RNAs regardless of the orientation of this fragment in the cloning vector DNA; thus, only the in vivo sense strand of the template is specifically transcribed in the in vitro system. The system is specific for the RNA polymerase responsible for the transcription of ribosomal RNA genes in vivo. Faithful transcription, like RNA polymerase I from Acanthamoeba, is insensitive to alpha-amanitin inhibition, and transcription is greatly stimulated by highly purified RNA polymerase I but not by RNA polymerases II or III. Conditions for optimal transcription were determined.

Faithful transcription of ribosomal 5-S RNA in vitro depends on the presence of several factors.

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5-S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G-100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5-S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5-S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 +/- 1 kDa stimulates the formation of 5-S RNA. This protein is contained in the breakthrough of DEAE-cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl. In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G-100 contains functional complexes, which are capable of transcribing the cloned 5-S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio-Gel A5m.

A stable transcription complex directs mouse ribosomal RNA synthesis by RNA polymerase I.

Ribosomal RNA is synthesized from template molecules that are activated by a stable association with essential transcription factors. This activated template assembles prior to the onset of transcription as a preinitiation complex and factors remain firmly attached during active elongation as well. Sequential addition of differently marked rRNA genes to an S-100 mouse cell extract shows that the DNA binding factors of the stable complex are present in limiting quantities. They associate rapidly with template molecules and the resultant transcription complex remains intact over prolonged periods of incubation in the presence of competitor DNA. The resistance of the stable complex to the usual inhibitory effect of high DNA concentration suggests that more than one DNA binding factor recognizes the rDNA promoter region and is needed to direct faithful transcription. Finally, although the stable complex is specific for the rRNA initiation region, added vector sequences can neutralize nonspecific DNA binding components that are also present in the cell extract. This lowers the requirement for rDNA template and demonstrates that each activated rRNA gene can direct at least 10 rounds of elongation and reinitiation.

The origin of the rRNA precursor from Xenopus borealis, analysed in vivo and in vitro.

We have determined the origin of the major transcript of Xenopus borealis rDNA by the use of an SI nuclease protection assay. The DNA surrounding the origin of this transcript was sequenced, and the region upstream of the origin was shown to have strong sequence homology with that region from X.laevis rDNA. We have also demonstrated faithful transcription from this origin using cloned X.borealis rDNA in an extract derived from X. laevis culture cells. This in vitro transcription was insensitive to 100 micrograms/ml alpha-amanatin, suggesting that it was mediated by RNA polymerase 1.

Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5′ flanking sequence far upstream

For the faithful initiation of transcription of exogenously added fibroin gene, a crude cellular extract was prepared from the posterior silk glands where fibroin gene is specifically transcribed. The faithful initiation was demonstrated by the assay with use of truncated fibroin gene templates and confirmed by nuclease S1 mapping. The transcription initiation of fibroin gene was detected also in the extract from the middle silk glands where fibroin gene is not transcribed, indicating that an expected suppression machinery for the gene was left behind during the extraction procedures. The promoter sequence of fibroin gene required in these homologous extracts (the nucleotide position −29 to +6) is the same as that in a heterologous HeLa cell extract. This result guarantees a limited usefulness of the HeLa cell extract in determining promoter sequence or analyzing general transcription machinery. In the homologous extracts, but not in the heterologous extract, however, it has been shown for the first time in vitro that the 5′ flanking sequence upstream from −74 enhances the transcription initiation of fibroin gene. In addition, a remarkable fibroin gene preference in transcription over mouse β globin and adenovirus 2 major late genes was observed in the homologous extracts. The fibroin gene preference is partly caused by the sequence element upstream from −74 of fibroin DNA.

Promoter sequence of fibroin gene assigned by in vitro transcription system.

We have shown that the silk fibroin gene from Bombyx mori is faithfully transcribed in an in vitro transcription system of the HeLa cell extract prepared by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Using this system and a series of deletion mutants of fibroin gene, we have assigned the promoter sequence of fibroin gene. The 5' boundary of the promoter is around nucleotide position -29, indicating that most of the T-A-T-A-A-A-A sequence (-30 to -24) is essential for the promoter function, where the transcription initiation point of fibroin gene is assigned as nucleotide position +1 [Tsuda, M., Ohshima, Y. & Suzuki, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 4872-4876]. The 3' boundary is around nucleotide position +6. However, to support the efficient, faithful transcription, some additional (more than 26 but less than 41) nucleotides of nonspecific origin are required at the 5' side of -29. Functions ascribed to the promoter region are discussed.

Control of Transcription of tRNA Genes

Our laboratory is interested in the regulation of gene expression during eukaryotic development. Among the genes we are currently studying are those coding for alanine tRNA in the silkworm, Bombyx mori. These genes are particularly interesting because one Bombyx alanine tRNA species is constitutive -- that is, it is found in a variety of cell types -- while the other major alanine tRNA appears only in the silkgland. The silkgland-specific tRNA predominates when the highly specialized cells of this tissue are synthesizing large amounts of the alanine-rich silk protein, fibroin. The nucleotide sequences of the two alanine tRNAs indicates that they are encoded by different genes.Our goal is to understand the molecular mechanisms governing the differential expression of Bombyx tRNAAla genes. Our approach is to isolate the genes with molecular cloning techniques, and to transcribe and process them in vitro. We have cloned 14 different genomic DNA fragments containing tRNAAla genes, and have developed faithful transcription systems from several different Bombyx tissues, including silkgland.