scholarly journals Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115 ◽  
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

scholarly journals Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551 ◽  
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-β subunit: influence of mutation on hCG production

1990 ◽  
Vol 5 (2) ◽  
pp. 97-102 ◽  
Author(s):  
C. Azuma ◽  
K. Miyai ◽  
F. Saji ◽  
S. Kamiura ◽  
Y. Tokugawa ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
In Vitro Mutagenesis ◽  
Β Subunit ◽  
Site Specific ◽  
Biological Specificity ◽  
Covalently Linked

ABSTRACT The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the α and β subunits. The β subunit is specific for each hormone and is responsible for the biological specificity, but the β subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different β subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-β subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the β subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.

Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

scholarly journals Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

2007 ◽  
Vol 189 (17) ◽  
pp. 6303-6311 ◽  
Author(s):  
Asa Flanigan ◽  
Jeffrey F. Gardner
Keyword(s):  
Dimethyl Sulfate ◽  
Direct Repeats ◽  
Mobility Shift ◽  
Base Pairs ◽  
Recombination Mechanism ◽  
Site Specific ◽  
Dnase I Footprinting ◽  
Pair Sequence ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

scholarly journals The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.

1990 ◽  
Vol 110 (4) ◽  
pp. 1199-1210 ◽  
Author(s):  
M Hatzfeld ◽  
K Weber
Keyword(s):  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Air Oxidation ◽  
Cross Link ◽  
Site Specific

Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine. The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross-link were obtained when filaments formed by the two reduced monomers were allowed to oxidize. Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed. Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer. This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins. In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates. However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation. Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation. Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link. This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.

The Hierarchic Order of Intermediate Filament Structure and Assembly

1985 ◽  
Vol 43 ◽  
pp. 722-725
Author(s):  
U. Aebi ◽  
E.C. Glavaris ◽  
R. Eichner
Keyword(s):  
Tissue Specificity ◽  
Structural Model ◽  
Eukaryotic Cells ◽  
Cdna Sequencing ◽  
Filament Structure ◽  
Keratin Filaments ◽  
Isoelectric Points ◽  
Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

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