title: The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

authors: Summers, Emma Louise

subjects: Science & Technology

abstract: Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNAbinding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal crosslinking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 A˚ resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß–ß–a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel b-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

text: The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNAbinding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal crosslinking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 A˚ resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß–ß–a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel b-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

year: 2012-06-13

title: Isolation and characterization of an RNA polymerase III encoded gene of Pinus radiata and its use in pine transformation

authors: Anandasayanan, M.

abstract: Several promoters such as the cauliflower mosaic virus 35S promoter (CaMV 35S) and its enhanced version, pEMU, the maize ubiquitin promoter, the alcohol dehydrogenase promoter (Adh) and rice actin promoter (Act1) are currently used in Pinus radiata (pine) transformation. These heterologous promoters were adopted for pine transformation for want of an endogenous promoter tailored specifically for the needs of pine. These promoters may not perform to the same extent in pine as in their homologous systems due to differences in quality and/or quantity of regulatory factors. Secondly, because of their heterologous origin, these promoters are open to silencing mechanisms that operate in plants against invasive DNA [Matzke & Birchler, 2005]. This could result in inactivation of these promoters at any time during the 30-year growth period of transformed pine which this poses a real threat to a forestry industry based on transgenic pine. 
A pine promoter on the other hand, being endogenous, is less prone to silencing. In addition, confidence in its longevity (continued expression) can be easily established even before using it in transformation. The aim of this study was to isolate and validate pine promoters that can be used in pine transformation. As only a few pine sequences were available in the public domain for gene discovery in pine (at the beginning of this study), heterologous sequence information was used to screen the pine genome or its transcriptome for orthologs with desirable expression features.
The investigation proceeded along two lines. In the first approach, a putatively desirable gene was isolated and the expression profile of its promoter was then validated. This led to the characterization of 5Spr20, a pine SS rDNA paralog. 5Spr20 differs from all published SS rDNA sequence& dnd is therefore a novel pine gene. Analyses of its sequence using bioinformatics revealed that it is capable of initiating biologically active transcripts and 5Spr20 is therefore a functional gene. A recombinant 5Spr20 promoter consisting of the coding region and the immediately upstream region downregulated gus reporter activity by 90% by antisense activity in transient expression studies in pine embryogenic cells. In stable expression studies, a 5Spr20 promoter-driven shDNA construct targeting gus completely silenced reporter activity in the model plant Nicotiana benthamiana. The 5Spr20 promoter appears to hold great promise for use in pine functional genomics and in gene downregulation applications. 
In the second line of investigation, the expression profiles of pine orthologs of known heterologous genes were validated prior to gene isolation. Two pine genes that were identified as promising candidates are pine tDNAMet-l and an actin paralog pine, ActX. Both genes were strongly expressed in all vegetative tissues of pine. Several PCR-based methods were used to clone the upstream regions (containing putative promoter elements) but all attempts ended in failure, which is attributed to the presence of pseudogenes and regions homologous to walking/sequencing primers among paralogs. 
The pine transcriptome was also screened unsuccessfully for ortholgs of desirable heterologous candidate genes like the ribosomal protein genes MsRL5 of Medicago sativa and AtL 18 of Arabidopsis thaliana and genes for the second largest subunit of RNA polymerase II, gene T13794 and actin-2 of A. thaliana. Sequence heterogeneity, cell-specific expression and low transcript abundance are possible reasons for not being able to detect pine orthologs of these candidate genes in expression screens.

text: Isolation and characterization of an RNA polymerase III encoded gene of Pinus radiata and its use in pine transformation Several promoters such as the cauliflower mosaic virus 35S promoter (CaMV 35S) and its enhanced version, pEMU, the maize ubiquitin promoter, the alcohol dehydrogenase promoter (Adh) and rice actin promoter (Act1) are currently used in Pinus radiata (pine) transformation. These heterologous promoters were adopted for pine transformation for want of an endogenous promoter tailored specifically for the needs of pine. These promoters may not perform to the same extent in pine as in their homologous systems due to differences in quality and/or quantity of regulatory factors. Secondly, because of their heterologous origin, these promoters are open to silencing mechanisms that operate in plants against invasive DNA [Matzke & Birchler, 2005]. This could result in inactivation of these promoters at any time during the 30-year growth period of transformed pine which this poses a real threat to a forestry industry based on transgenic pine. 
A pine promoter on the other hand, being endogenous, is less prone to silencing. In addition, confidence in its longevity (continued expression) can be easily established even before using it in transformation. The aim of this study was to isolate and validate pine promoters that can be used in pine transformation. As only a few pine sequences were available in the public domain for gene discovery in pine (at the beginning of this study), heterologous sequence information was used to screen the pine genome or its transcriptome for orthologs with desirable expression features.
The investigation proceeded along two lines. In the first approach, a putatively desirable gene was isolated and the expression profile of its promoter was then validated. This led to the characterization of 5Spr20, a pine SS rDNA paralog. 5Spr20 differs from all published SS rDNA sequence& dnd is therefore a novel pine gene. Analyses of its sequence using bioinformatics revealed that it is capable of initiating biologically active transcripts and 5Spr20 is therefore a functional gene. A recombinant 5Spr20 promoter consisting of the coding region and the immediately upstream region downregulated gus reporter activity by 90% by antisense activity in transient expression studies in pine embryogenic cells. In stable expression studies, a 5Spr20 promoter-driven shDNA construct targeting gus completely silenced reporter activity in the model plant Nicotiana benthamiana. The 5Spr20 promoter appears to hold great promise for use in pine functional genomics and in gene downregulation applications. 
In the second line of investigation, the expression profiles of pine orthologs of known heterologous genes were validated prior to gene isolation. Two pine genes that were identified as promising candidates are pine tDNAMet-l and an actin paralog pine, ActX. Both genes were strongly expressed in all vegetative tissues of pine. Several PCR-based methods were used to clone the upstream regions (containing putative promoter elements) but all attempts ended in failure, which is attributed to the presence of pseudogenes and regions homologous to walking/sequencing primers among paralogs. 
The pine transcriptome was also screened unsuccessfully for ortholgs of desirable heterologous candidate genes like the ribosomal protein genes MsRL5 of Medicago sativa and AtL 18 of Arabidopsis thaliana and genes for the second largest subunit of RNA polymerase II, gene T13794 and actin-2 of A. thaliana. Sequence heterogeneity, cell-specific expression and low transcript abundance are possible reasons for not being able to detect pine orthologs of these candidate genes in expression screens.

year: 2006

title: Perception and flagging of loanwords – A diachronic case-study of Māori loanwords in New Zealand English

authors: Levendis, Katharine

abstract: This paper combines a quantitative and qualitative analysis of a diachronic corpus of New Zealand newspapers built to analyse the use of Māori loanwords in New Zealand English. We report findings in relation to flagging (marking of loanwords as being foreign material in a given language) and show that it is (weakly) predicated by frequency-of-use and by semantic category of the loanword (core loans are flagged more than cultural ones), but not by listedness. Alongside this trend, we note that perceptions of writers using the words vary enormously in regard to which loans are integrated and familiar, matching neither listedness nor frequency-of-use patterns. This indicates that in NZE, loanword use remains strongly tied up with socio-political identity and language ideology, rather than rooted in linguistic factors (such as, bilingualism or filling in lexical gaps).

text: Perception and flagging of loanwords – A diachronic case-study of Māori loanwords in New Zealand English This paper combines a quantitative and qualitative analysis of a diachronic corpus of New Zealand newspapers built to analyse the use of Māori loanwords in New Zealand English. We report findings in relation to flagging (marking of loanwords as being foreign material in a given language) and show that it is (weakly) predicated by frequency-of-use and by semantic category of the loanword (core loans are flagged more than cultural ones), but not by listedness. Alongside this trend, we note that perceptions of writers using the words vary enormously in regard to which loans are integrated and familiar, matching neither listedness nor frequency-of-use patterns. This indicates that in NZE, loanword use remains strongly tied up with socio-political identity and language ideology, rather than rooted in linguistic factors (such as, bilingualism or filling in lexical gaps).

year: 2019