scholarly journals Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2971
Author(s):  
Tatsuji Hataya ◽  
Takashi Naoi
Keyword(s):  
Transcription Initiation ◽  
Potato Spindle Tuber Viroid ◽  
Initiation Site ◽  
Cdna Clones ◽  
In Vitro Mutagenesis ◽  
Replication Process ◽  
Rna Transcripts ◽  
Terminal Structure ◽  
Ligation Site

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 3067-3077 ◽  
Author(s):  
J.S. Margolis ◽  
M.L. Borowsky ◽  
E. Steingrimsson ◽  
C.W. Shim ◽  
J.A. Lengyel ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Embryo ◽  
Upstream Region ◽  
Enhancer Element ◽  
Gap Gene ◽  
Gap Genes ◽  
Two Phases ◽  
Necessary And Sufficient

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro

1985 ◽  
Vol 5 (2) ◽  
pp. 380-389
Author(s):  
S M Hanly ◽  
G C Bleecker ◽  
N Heintz
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
S Phase ◽  
Histone H4 ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Distal Promoter ◽  
Histone H4 Gene

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA

1990 ◽  
Vol 10 (5) ◽  
pp. 2315-2326
Author(s):  
J H Teruya ◽  
S Y Kutsunai ◽  
D H Spear ◽  
P A Edwards ◽  
C F Clarke
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Farnesyl Pyrophosphate ◽  
S1 Nuclease ◽  
Testis Cdna ◽  
Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV

2005 ◽  
Vol 86 (9) ◽  
pp. 2585-2593 ◽  
Author(s):  
F. F. Huang ◽  
F. W. Pierson ◽  
T. E. Toth ◽  
X. J. Meng
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Cdna Clones ◽  
Virus Shedding ◽  
Rna Transcripts ◽  
Successful Infection ◽  
The Usa ◽  
Avian Hev

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis–splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

scholarly journals Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA.

1990 ◽  
Vol 10 (5) ◽  
pp. 2315-2326 ◽  
Author(s):  
J H Teruya ◽  
S Y Kutsunai ◽  
D H Spear ◽  
P A Edwards ◽  
C F Clarke
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Farnesyl Pyrophosphate ◽  
S1 Nuclease ◽  
Testis Cdna ◽  
Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

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