In vitro
 recapitulation of the site-specific editing (to wild-type) of mutant IDS
 mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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A Point Mutation in the mma3 Gene Is Responsible for Impaired Methoxymycolic Acid Production in Mycobacterium bovis BCG Strains Obtained after 1927

Journal of Bacteriology ◽

10.1128/jb.182.12.3394-3399.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3394-3399 ◽

Cited By ~ 59

Author(s):

Marcel A. Behr ◽

Benjamin G. Schroeder ◽

Jacquelyn N. Brinkman ◽

Richard A. Slayden ◽

Clifton E. Barry

Keyword(s):

Point Mutation ◽

Mycobacterium Bovis ◽

Mycobacterium Smegmatis ◽

Pasteur Institute ◽

Wild Type ◽

Phenotypic Properties ◽

Site Specific ◽

Mutant Population ◽

Mycolic Acids

ABSTRACT BCG vaccines are substrains of Mycobacterium bovisderived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production andmma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, themma3 sequence of the latter strains differs from that ofMycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

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Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

Biochemical Journal ◽

10.1042/bj20080881 ◽

2008 ◽

Vol 415 (1) ◽

pp. 35-43 ◽

Cited By ~ 23

Author(s):

Jens F. Rehfeld ◽

Xiaorong Zhu ◽

Christina Norrbom ◽

Jens R. Bundgaard ◽

Anders H. Johnsen ◽

...

Keyword(s):

Structural Factors ◽

Cleavage Sites ◽

Wild Type ◽

Prohormone Convertases ◽

Site Specific ◽

G Cells ◽

Processing Site ◽

Vitro Effect

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg36Arg37 and Arg73Arg74 sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, α-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys53Lys54), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

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Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

Canadian Journal of Genetics and Cytology ◽

10.1139/g84-060 ◽

1984 ◽

Vol 26 (3) ◽

pp. 386-389 ◽

Cited By ~ 1

Author(s):

Linda J. Reha-Krantz ◽

Sükran Parmaksizoglu

Keyword(s):

Dna Polymerase ◽

Low Temperatures ◽

Bacteriophage T4 ◽

Effect Of Temperature ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

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Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

Journal of Endocrinology ◽

10.1677/joe.1.06939 ◽

2006 ◽

Vol 191 (1) ◽

pp. 101-111 ◽

Cited By ~ 39

Author(s):

David J Flint ◽

Nadine Binart ◽

Stephanie Boumard ◽

John J Kopchick ◽

Paul Kelly

Keyword(s):

Adipose Tissue ◽

Receptor Gene ◽

Metabolic Effects ◽

Wild Type ◽

Extrinsic Factors ◽

Differentiation Potential ◽

Site Specific ◽

Proliferation And Differentiation

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40–50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

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Characterization of Holliday structures in FLP protein-promoted site-specific recombination

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.235-242.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 235-242

Author(s):

L Meyer-Leon ◽

R B Inman ◽

M M Cox

Keyword(s):

Reaction Rate ◽

Reaction Pathway ◽

Reaction Intermediates ◽

Wild Type ◽

Site Specific ◽

Site Specific Recombination ◽

Isomerization Process ◽

Previous Demonstration ◽

Rate Limiting

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

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Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

Visual Neuroscience ◽

10.1017/s0952523812000223 ◽

2012 ◽

Vol 29 (4-5) ◽

pp. 211-217 ◽

Cited By ~ 18

Author(s):

CONSTANZE BICKELMANN ◽

JAMES M. MORROW ◽

JOHANNES MÜLLER ◽

BELINDA S.W. CHANG

Keyword(s):

Half Life ◽

Functional Characterization ◽

Egg Laying ◽

Sensory Transduction ◽

Wild Type ◽

Functional Variation ◽

Bovine Rhodopsin ◽

Tachyglossus Aculeatus

AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

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Purification and Characterization of the R64 Shufflon-Specific Recombinase

Journal of Bacteriology ◽

10.1128/jb.182.10.2787-2792.2000 ◽

2000 ◽

Vol 182 (10) ◽

pp. 2787-2792 ◽

Cited By ~ 14

Author(s):

Atsuko Gyohda ◽

Teruya Komano

Keyword(s):

Electrophoretic Analysis ◽

Recombination Reaction ◽

Site Specific ◽

Dna Inversion ◽

Repeat Sequences ◽

In Vitro Recombination ◽

Recombinase Gene ◽

A Site

ABSTRACT The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

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