Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

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RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2530-2537.1987 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2530-2537 ◽

Cited By ~ 1

Author(s):

H Zhu ◽

I G Macreadie ◽

R A Butow

Keyword(s):

Rna Processing ◽

Rrna Gene ◽

Yeast Mitochondria ◽

Wild Type ◽

Reading Frame ◽

Processing Activity ◽

Novel Processing ◽

Type Strains ◽

Upstream Exon

The 3' ends of most Saccharomyces cerevisiae mitochondrial mRNAs terminate at a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3', of unknown function. We have studied the consequences of mutations within a dodecamer found in an 1,143-base-pair optional intron of the mitochondrial large (21S) rRNA gene on RNA processing. The dodecamer is situated at the 3' end of an expressed open reading frame (ORF) within that intron, and the mutations are two adjacent transversions that extend the intron ORF by 51 nucleotides. The strain harboring these mutations, L5-10-1, is defective in biased intron transmission in crosses to strains that lack the intron, as are other mutants which contain nucleotide changes within the ORF (I. G. Macreadie, R. M. Scott, A. R. Zinn, and R. A. Butow, Cell 41:395-402, 1985). However, unlike these other mutants, wild-type strains, or petites which retain the intron allele, L5-10-1 is defective in processing at the intron dodecamer. In addition, L5-10-1 lacks a prominent 2.7-kilobase RNA containing both intron and exon sequences and at least two of four RNAs that correspond to various forms of the excised intron. We propose that these RNAs, missing in L5-10-1 but present in all other strains examined, arise in part by processing at the intron dodecamer. In addition, in all strains examined, we have detected a novel processing activity in which precursor 21S rRNA transcripts are cleaved in the upstream exon, about 1,500 nucleotides from the 5' end of the RNA. This activity, together with 3' intron dodecamer cleavage, probably accounts for the 2.7-kilobase RNA species, a candidate for the mRNA for the intron-encoded protein.

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RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2530 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2530-2537 ◽

Cited By ~ 14

Author(s):

H Zhu ◽

I G Macreadie ◽

R A Butow

Keyword(s):

Rna Processing ◽

Rrna Gene ◽

Yeast Mitochondria ◽

Wild Type ◽

Reading Frame ◽

Processing Activity ◽

Novel Processing ◽

Type Strains ◽

Upstream Exon

The 3' ends of most Saccharomyces cerevisiae mitochondrial mRNAs terminate at a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3', of unknown function. We have studied the consequences of mutations within a dodecamer found in an 1,143-base-pair optional intron of the mitochondrial large (21S) rRNA gene on RNA processing. The dodecamer is situated at the 3' end of an expressed open reading frame (ORF) within that intron, and the mutations are two adjacent transversions that extend the intron ORF by 51 nucleotides. The strain harboring these mutations, L5-10-1, is defective in biased intron transmission in crosses to strains that lack the intron, as are other mutants which contain nucleotide changes within the ORF (I. G. Macreadie, R. M. Scott, A. R. Zinn, and R. A. Butow, Cell 41:395-402, 1985). However, unlike these other mutants, wild-type strains, or petites which retain the intron allele, L5-10-1 is defective in processing at the intron dodecamer. In addition, L5-10-1 lacks a prominent 2.7-kilobase RNA containing both intron and exon sequences and at least two of four RNAs that correspond to various forms of the excised intron. We propose that these RNAs, missing in L5-10-1 but present in all other strains examined, arise in part by processing at the intron dodecamer. In addition, in all strains examined, we have detected a novel processing activity in which precursor 21S rRNA transcripts are cleaved in the upstream exon, about 1,500 nucleotides from the 5' end of the RNA. This activity, together with 3' intron dodecamer cleavage, probably accounts for the 2.7-kilobase RNA species, a candidate for the mRNA for the intron-encoded protein.

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The role of a conserved dodecamer sequence in yeast mitochondrial gene expression

Genome ◽

10.1139/g89-134 ◽

1989 ◽

Vol 31 (2) ◽

pp. 757-760 ◽

Cited By ~ 28

Author(s):

Ronald A. Butow ◽

Hong Zhu ◽

Philip Perlman ◽

Heather Conrad-Webb

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Mitochondrial Gene ◽

Rna Stability ◽

Rrna Gene ◽

Mitochondrial Gene Expression ◽

Reading Frame ◽

Multicomponent Complex ◽

Var1 Gene

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5′-AAUAAUAUUCUU-3′. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the ω+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.Key words: mitochondria, yeast, mRNA, RNA processing, 3′ dodecamer.

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Genetic Requirements for Salmonella-Induced Cytopathology in Human Monocyte-Derived Macrophages

Infection and Immunity ◽

10.1128/iai.70.12.7126-7135.2002 ◽

2002 ◽

Vol 70 (12) ◽

pp. 7126-7135 ◽

Cited By ~ 42

Author(s):

Sara H. Browne ◽

Marc L. Lesnick ◽

Donald G. Guiney

Keyword(s):

Salmonella Enterica ◽

Human Monocyte ◽

Intracellular Bacteria ◽

Wild Type ◽

Site Specific ◽

Human Macrophages ◽

Serovar Typhimurium ◽

Protein Secretion System ◽

Type Iii Protein Secretion ◽

A Site

ABSTRACT Infection of human macrophages with Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Dublin produces delayed cytotoxicity characterized by cell detachment and associated apoptosis. Using a site-specific mutant in the SpvB active site, we verify that the ADP-ribosylation activity of SpvB is required for delayed cytotoxicity in human macrophages infected with Salmonella. SipB and the type III protein secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) are not involved, whereas the SPI2 TTSS is absolutely required for SpvB-dependent cytotoxicity. Furthermore, we show that infection of macrophage cultures with wild-type or sipB mutant bacteria led to a complete loss of polymerized actin in over half of the cells after 24 h. In contrast, macrophages infected with the spvB or SPI2 (ssaV or ssaJ) mutant strain retained normal F-actin filaments, despite similar numbers of intracellular bacteria. We conclude that SpvB and a functional SPI2 TTSS are essential for Salmonella-induced delayed cytotoxicity of human macrophages.

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Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation.

Journal of Virology ◽

10.1128/jvi.67.12.7223-7228.1993 ◽

1993 ◽

Vol 67 (12) ◽

pp. 7223-7228 ◽

Cited By ~ 54

Author(s):

E K Subbarao ◽

Y Kawaoka ◽

B R Murphy

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Temperature Sensitive ◽

Wild Type ◽

Site Specific ◽

Mutant Derivative ◽

Specific Temperature ◽

A Site

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Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

Biochemical Journal ◽

10.1042/bj20080668 ◽

2008 ◽

Vol 414 (2) ◽

pp. 205-214 ◽

Cited By ~ 51

Author(s):

Oliver Mueller-Cajar ◽

Spencer M. Whitney

Keyword(s):

Escherichia Coli ◽

Large Subunit ◽

Fold Increase ◽

Functional Expression ◽

Kinetic Properties ◽

Selection System ◽

Wild Type ◽

Bisphosphate Carboxylase ◽

E Coli ◽

Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

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Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2087-2096.1987 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2087-2096

Author(s):

B Sauer

Keyword(s):

Saccharomyces Cerevisiae ◽

Functional Expression ◽

Yeast Saccharomyces Cerevisiae ◽

Efficient Manner ◽

Site Specific ◽

Site Specific Recombination ◽

Leu2 Gene ◽

Recombination System ◽

A Site ◽

Site Specific Recombination System

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

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The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

Applied and Environmental Microbiology ◽

10.1128/aem.00207-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 5

Author(s):

Coral González-Prieto ◽

Richard Gabriel ◽

Christoph Dehio ◽

Manfred Schmidt ◽

Matxalen Llosa

Keyword(s):

Human Genome ◽

Fold Increase ◽

Human Cells ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Dna Complexes ◽

Site Specific ◽

Type Iv ◽

Site Specific Integration ◽

A Site

ABSTRACT Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

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Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Journal of Bacteriology ◽

10.1128/jb.01537-08 ◽

2009 ◽

Vol 191 (6) ◽

pp. 1933-1940 ◽

Cited By ~ 2

Author(s):

André Larouche ◽

Paul H. Roy

Keyword(s):

Antibiotic Resistance Genes ◽

Cysteine Residue ◽

Wild Type ◽

Recombination Mechanism ◽

Integrase Gene ◽

Site Specific ◽

Site Specific Recombination ◽

Environmental Bacterium ◽

Target Sites ◽

A Site

ABSTRACT Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

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