Cis-Acting Ribozymes for the Production of RNA In Vitro Transcripts with Defined 5′ and 3′ Ends

2012 ◽  
pp. 83-98 ◽  
Author(s):  
Johanna M. Avis ◽  
Graeme L. Conn ◽  
Scott C. Walker
Keyword(s):  
Cis Acting ◽  
In Vitro Transcripts

Regulation of proboscipedia in Drosophila by Homeotic Selector Genes

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 183-194
Author(s):  
Douglas B Rusch ◽  
Thomas C Kaufman
Keyword(s):  
Expression Pattern ◽  
Regulatory Element ◽  
Polycomb Group ◽  
Accumulation Pattern ◽  
Cis Acting ◽  
Polycomb Group Genes ◽  
Gap Genes ◽  
Combined Action ◽  
Selector Genes

Abstract The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. We have identified several of the genes necessary to generate this embryonic pattern of expression. These genes can be roughly split into three categories based on their time of action during development. First, prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. Second, the initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). Lastly, maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. We further show that Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila melanogaster and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct.

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

scholarly journals Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

2002 ◽  
Vol 22 (19) ◽  
pp. 6726-6734 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Junichi Obokata ◽  
Masahiro Sugiura
Keyword(s):  
Rna Editing ◽  
Editing Site ◽  
Mrna Editing ◽  
Higher Plant ◽  
In Vitro System ◽  
Cis Acting ◽  
Biochemical Identification ◽  
Editing Activity

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

scholarly journals In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

2021 ◽  
Author(s):  
Fee Zimmermann ◽  
Maria Urban ◽  
Christian Krueger ◽  
Mathias Walter ◽  
Roman Woelfel ◽  
...  
Keyword(s):  
Binding Sites ◽  
Detrimental Effect ◽  
In Vitro Evaluation ◽  
In Vitro Transcripts ◽  
Nucleotide Mismatch ◽  
Reverse Primer ◽  
Forward Primer

A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charite RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.

Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor

1988 ◽  
Vol 8 (6) ◽  
pp. 2572-2580
Author(s):  
A Goel ◽  
R E Pearlman
Keyword(s):  
Gene Expression ◽  
Base Pair ◽  
Specific Binding ◽  
Adjacent Gene ◽  
Cis Acting ◽  
Contact Sites ◽  
Cell Extracts ◽  
Single Base Pair ◽  
Beta Galactosidase

In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

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