Donna Esposito - Academia.edu (original) (raw)

Papers by Donna Esposito

Initiation codon context is an important determinant of translation initiation rates in both prok... more Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAf Met anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA fMet. The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions.

The EMBO Journal, 2003

Initiation codon context is an important determinant of translation initiation rates in both prok... more Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAf Met anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA fMet. The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions.

Molecular Microbiology, 2005

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a proces... more Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli , retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonucleasedependent pathway. However, in retrotransposition in L. lactis , the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonucleaseindependent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli , using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis , in E. coli , Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonucleaseindependent pathway, and, as in L. lactis , such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.

Molecular and General Genetics MGG, 2000

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP... more The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.

THE PLANT CELL ONLINE, 2001

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the th... more To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the ؊ 1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the ؊ 1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.

A nucleus-encoded suppressor de®nes a new factor which can promote petD mRNA stability in the chl... more A nucleus-encoded suppressor de®nes a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii

Journal of Immunology, 2013

SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoA... more SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoAbs to acetylcholine receptors, detectable in humans, impair salivation in SS-afflicted mice with NOD Aec2 and Aec1 susceptibility loci. COX-2 is a potential target for reducing SS pathogenic IgG Ab and B cell lymphoma, since (a) within in vitro cultures B cell-expressed COX-2 and PGE2 augment DNA-modifying AID and downstream IgG switching and (b) mice deficient in COX-2 have reduced IgG. If B cell specific COX-2 function is critical for SS disease, cell-targeted delivery of inhibitors might circumvent adverse side effects. With a downstream goal of examining whether B cell COX-2 contributes to autoimmunity, we introduced a floxed COX-2 gene and a CD19Cre gene independently onto the NOD.B10-H2b SS-susceptible background. To our surprise, early congenic lines HET for either CD19Cre or COX-2flox failed to show aging-related hyposalivation (xerostomia), in contrast to wild-type NOD.B10. Redu...

Clinical Immunology, 2014

Sjogren's syndrome (SS) is characterized by salivary glan... more Sjogren's syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.

Current Genetics

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chlorop... more Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b 6/f complex. The MCD1 gene product is thought to interact with the mRNA 5′ end to protect it from degradation by a 5′ → 3′ exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability...

Initiation codon context is an important determinant of translation initiation rates in both prok... more Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAf Met anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA fMet. The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions.

The EMBO Journal, 2003

Initiation codon context is an important determinant of translation initiation rates in both prok... more Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAf Met anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA fMet. The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions.

Molecular Microbiology, 2005

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a proces... more Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli , retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonucleasedependent pathway. However, in retrotransposition in L. lactis , the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonucleaseindependent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli , using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis , in E. coli , Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonucleaseindependent pathway, and, as in L. lactis , such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.

Molecular and General Genetics MGG, 2000

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP... more The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.

THE PLANT CELL ONLINE, 2001

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the th... more To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the ؊ 1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the ؊ 1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.

A nucleus-encoded suppressor de®nes a new factor which can promote petD mRNA stability in the chl... more A nucleus-encoded suppressor de®nes a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii

Journal of Immunology, 2013

SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoA... more SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoAbs to acetylcholine receptors, detectable in humans, impair salivation in SS-afflicted mice with NOD Aec2 and Aec1 susceptibility loci. COX-2 is a potential target for reducing SS pathogenic IgG Ab and B cell lymphoma, since (a) within in vitro cultures B cell-expressed COX-2 and PGE2 augment DNA-modifying AID and downstream IgG switching and (b) mice deficient in COX-2 have reduced IgG. If B cell specific COX-2 function is critical for SS disease, cell-targeted delivery of inhibitors might circumvent adverse side effects. With a downstream goal of examining whether B cell COX-2 contributes to autoimmunity, we introduced a floxed COX-2 gene and a CD19Cre gene independently onto the NOD.B10-H2b SS-susceptible background. To our surprise, early congenic lines HET for either CD19Cre or COX-2flox failed to show aging-related hyposalivation (xerostomia), in contrast to wild-type NOD.B10. Redu...

Clinical Immunology, 2014

Sjogren's syndrome (SS) is characterized by salivary glan... more Sjogren's syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.

Current Genetics

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chlorop... more Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b 6/f complex. The MCD1 gene product is thought to interact with the mRNA 5′ end to protect it from degradation by a 5′ → 3′ exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability...