scholarly journals Assessing nucleic acid binding activity of four dinoflagellate cold shock domain proteins from Symbiodinium kawagutii and Lingulodinium polyedra

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Bahareh Zaheri ◽  
David Morse
Keyword(s):  
Transcription Factors ◽  
Nucleic Acid ◽  
Transcriptional Control ◽  
Cold Shock ◽  
Binding Activity ◽  
Dependent Manner ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Double Stranded Dna ◽  
Cold Shock Domain

Abstract Background Dinoflagellates have a generally large number of genes but only a small percentage of these are annotated as transcription factors. Cold shock domain (CSD) containing proteins (CSPs) account for roughly 60% of these. CSDs are not prevalent in other eukaryotic lineages, perhaps suggesting a lineage-specific expansion of this type of transcription factors in dinoflagellates, but there is little experimental data to support a role for dinoflagellate CSPs as transcription factors. Here we evaluate the hypothesis that dinoflagellate CSPs can act as transcription factors by binding double-stranded DNA in a sequence dependent manner. Results We find that both electrophoretic mobility shift assay (EMSA) competition experiments and selection and amplification binding (SAAB) assays indicate binding is not sequence specific for four different CSPs from two dinoflagellate species. Competition experiments indicate all four CSPs bind to RNA better than double-stranded DNA. Conclusion Dinoflagellate CSPs do not share the nucleic acid binding properties expected for them to function as bone fide transcription factors. We conclude the transcription factor complement of dinoflagellates is even smaller than previously thought suggesting that dinoflagellates have a reduced dependance on transcriptional control compared to other eukaryotes.

scholarly journals Characterization of Two Dinoflagellate Cold Shock Domain Proteins

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Mathieu Beauchemin ◽  
Sougata Roy ◽  
Sarah Pelletier ◽  
Alexandra Averback ◽  
Frederic Lanthier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Control ◽  
Cold Shock ◽  
Mobility Shift ◽  
Two Dimensional Gel Electrophoresis ◽  
Content Type ◽  
Cold Shock Domain ◽  
Cold Shock Domain Proteins ◽  
Specific Sequences

ABSTRACT Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists. Roughly two-thirds of the proteins annotated as transcription factors in dinoflagellate transcriptomes are cold shock domain-containing proteins (CSPs), an uncommon condition in eukaryotic organisms. However, no functional analysis has ever been reported for a dinoflagellate CSP, and so it is not known if they do in fact act as transcription factors. We describe here some of the properties of two CSPs from the dinoflagellate Lingulodinium polyedrum, LpCSP1 and LpCSP2, which contain a glycine-rich C-terminal domain and an N-terminal cold shock domain phylogenetically related to those in bacteria. However, neither of the two LpCSPs act like the bacterial CSP, since they do not functionally complement the Escherichia coli quadruple cold shock domain protein mutant BX04, and cold shock does not induce LpCSP1 and LpCSP2 to detectable levels, based on two-dimensional gel electrophoresis. Both CSPs bind to RNA and single-stranded DNA in a nonspecific manner in electrophoretic mobility shift assays, and both proteins also bind double-stranded DNA nonspecifically, albeit more weakly. These CSPs are thus unlikely to act alone as sequence-specific transcription factors. IMPORTANCE Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists.

scholarly journals A Novel Cold-Regulated Cold Shock Domain Containing Protein from Scallop Chlamys farreri with Nucleic Acid-Binding Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e32012 ◽  
Author(s):  
Chuanyan Yang ◽  
Lingling Wang ◽  
Vinu S. Siva ◽  
Xiaowei Shi ◽  
Qiufen Jiang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cold Shock ◽  
Binding Activity ◽  
Chlamys Farreri ◽  
Nucleic Acid Binding ◽  
Cold Shock Domain ◽  
Nucleic Acid Binding Activity

scholarly journals The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

1998 ◽  
Vol 18 (6) ◽  
pp. 3580-3585 ◽  
Author(s):  
Wenjin Zheng ◽  
Stephen Albert Johnston
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Dual Function ◽  
Nucleic Acid Binding ◽  
Peptide Cleavage ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Bleomycin Hydrolase ◽  
Nucleic Acid Binding Activity ◽  
Rna And Dna

ABSTRACT Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked double-stranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.

scholarly journals Nucleic Acid Binding Activity of Pns6 Encoded by Genome Segment 6 of Rice Ragged Stunt Oryzavirus

2004 ◽  
Vol 36 (7) ◽  
pp. 457-466 ◽  
Author(s):  
Chao-Gang Shao ◽  
Hui-Juan Lü ◽  
Jian-Hua Wu ◽  
Zu-Xun Gong
Keyword(s):  
Nucleic Acid ◽  
Western Blotting ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Genome Segment ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Mobility Shift ◽  
Dna And Rna ◽  
Nucleic Acid Binding Activity

Abstract The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single-and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.

scholarly journals Identification and Transcriptional Control of Caulobacter crescentus Genes Encoding Proteins Containing a Cold Shock Domain

2004 ◽  
Vol 186 (17) ◽  
pp. 5603-5613 ◽  
Author(s):  
Elza A. S. Lang ◽  
Marilis V. Marques
Keyword(s):  
Transcriptional Control ◽  
Cold Shock ◽  
Caulobacter Crescentus ◽  
Transcriptional Level ◽  
Deletion Mapping ◽  
Nucleic Acid Binding ◽  
Cold Shock Proteins ◽  
Reverse Transcription Pcr ◽  
Cold Shock Domain ◽  
Shock Proteins

ABSTRACT The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of α-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10°C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals 1P123 DMA binding analyses of bHLH transcription factors by FRET measurements(Nucleic acid-binding proteins,Poster Presentations)

2007 ◽  
Vol 47 (supplement) ◽  
pp. S54
Author(s):  
Koji HASEGAWA ◽  
Tatsushi GOTO ◽  
Daisuke KITANO ◽  
Mari KOTOURA ◽  
Fumio TOKUNAGA ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nucleic Acid ◽  
Binding Proteins ◽  
Nucleic Acid Binding ◽  
Nucleic Acid Binding Proteins ◽  
Poster Presentations ◽  
Bhlh Transcription Factors
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