The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin-deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green alga. Here we show that intron-containing gene fragments directly linked to their intron-less antisense counterpart provide efficient post-transcriptional gene silencing (PTGS) in C. reinhardtii, thus allowing an efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.

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Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya

Journal of General Virology ◽

10.1099/0022-1317-82-11-2827 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2827-2836 ◽

Cited By ~ 32

Author(s):

Chu-Hui Chiang ◽

Ju-Jung Wang ◽

Fuh-Jyh Jan ◽

Shyi-Dong Yeh ◽

Dennis Gonsalves

Keyword(s):

Coat Protein ◽

Sequence Similarity ◽

Transcriptional Gene Silencing ◽

Papaya Ringspot Virus ◽

Wild Type ◽

Coding Region ◽

Transgenic Papaya ◽

Cp Gene ◽

Post Transcriptional Gene Silencing

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5′ region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3′ parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3′-end regions of the CP gene and the 3′ non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.

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An historical and genomic view of schistosome conjugal biology with emphasis on sex-specific gene expression

Parasitology ◽

10.1017/s0031182004006213 ◽

2004 ◽

Vol 128 (S1) ◽

pp. S11-S22 ◽

Cited By ~ 21

Author(s):

K. F. HOFFMANN

Keyword(s):

Dna Microarrays ◽

Historical Review ◽

Transcriptional Gene Silencing ◽

Specific Gene ◽

Post Transcriptional Gene Silencing ◽

Genomic Tools ◽

Tools And Techniques ◽

Sexually Mature ◽

Identification And Characterization

The genetic programmes associated with the sexual biology of dioecious schistosomes remain a critically important but significantly understudied area of parasitology. Throughout the last four decades, progress has been slow in describing the gross antigenic and proteomic differences linked to sexually mature schistosomes and in characterizing some of the sex-associated transcripts and regulatory mechanisms induced during developmental maturation. These investigations have been severely hindered by the lack of complete EST/genomic information, as well as corresponding post- and functional-genomic tools for studying these pathogenic parasites. As near complete transcriptomes forSchistosoma japonicumandS. mansonihave recently been reported, and both DNA microarrays and post-transcriptional gene silencing have been applied to schistosomes, the tools and techniques for the high-throughput identification and characterization of transcripts involved in conjugal biology are now readily available. Here, an historical review is presented that summarizes some of the most significant findings associated with schistosome sex and sexual maturation during the last several decades. Following this discussion is a current overview of some modern day genomic approaches used to study schistosomes, which illustrates how major advances in the field of conjugal biology will be achieved.

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Dual-layer transposon repression in heads of Drosophila melanogaster

10.1101/267039 ◽

2018 ◽

Author(s):

Marius van den Beek ◽

Bruno da Silva ◽

Juliette Pouch ◽

Mohammed el amine Ali Chaouche ◽

Clément Carré ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Gene Silencing ◽

Rna Extraction ◽

Transcript Abundance ◽

Transcriptional Gene Silencing ◽

Loss Of Function ◽

Wild Type ◽

Rna Molecules ◽

Post Transcriptional Gene Silencing ◽

Ping Pong

AbstractpiRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of heritable mutations caused by their transposition in the genome. It is not clear whether the piRNA pathway plays a functional role in adult, non-gonadal tissues in Drosophila melanogaster. To address this question, we first analyzed the small RNA content of adult Drosophila melanogaster heads. We found that varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of piRNAs detected in head sequencing libraries originate from gonads. We next sequenced the heads of wild type and piwi mutants to address whether piwi loss of function would affect the low amount of piRNA-sized, ping-pong negative molecules that are still detected in heads hand-checked to avoid gonadal contamination. We find that loss of piwi does not affect significantly these 24-28 RNA molecules. Instead, we observe increased siRNA levels against the majority of Drosophila transposable element families. To determine the effect of this siRNA level change on transposon expression, we sequenced the transcriptome of wild type, piwi, dicer-2 and piwi, dicer-2 double-mutant fly heads. We find that RNA expression levels of the majority of TE families in piwi or dicer-2 mutants remain unchanged and that TE transcript abundance increases significantly only in piwi, dicer-2 double-mutants. These results lead us to suggest a dual-layer model for TE repression in adult somatic tissues. Piwi-mediated transcriptional gene silencing (TGS) established during embryogenesis constitutes the first layer of TE repression whereas Dicer-2-dependent siRNA-mediated post-transcriptional gene silencing (PTGS) provide a backup mechanism to repress TEs that escape silencing by piwi-mediated TGS.

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Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization

The Journal of Cell Biology ◽

10.1083/jcb.201009131 ◽

2011 ◽

Vol 193 (4) ◽

pp. 741-753 ◽

Cited By ~ 30

Author(s):

Telsa M. Mittelmeier ◽

Joseph S. Boyd ◽

Mary Rose Lamb ◽

Carol L. Dieckmann

Keyword(s):

Chlamydomonas Reinhardtii ◽

Green Alga ◽

Basal Body ◽

Cytoskeletal Protein ◽

Wild Type ◽

Unicellular Green Alga ◽

Protein Mutations ◽

The Relationship ◽

Mutant Cells ◽

In Cells

The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells.

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Sequence-, Tissue-, and Delivery-Specific Targeting of RNA During Post-Transcriptional Gene Silencing

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.8.753 ◽

2002 ◽

Vol 15 (8) ◽

pp. 753-763 ◽

Cited By ~ 6

Author(s):

Ezequiel Balmori-Melian ◽

Robin M. MacDiarmid ◽

David L. Beck ◽

Richard C. Gardner ◽

Richard L. S. Forster

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Viral Vectors ◽

Transcriptional Gene Silencing ◽

Wild Type ◽

Long Distance ◽

Post Transcriptional Gene Silencing ◽

In The Wild ◽

Long Distance Movement ◽

White Clover Mosaic Virus

Transgenic Nicotiana benthamiana plants expressing an untranslatable version of the coat protein (CP) gene from the Tamarillo mosaic virus (TaMV) were either resistant to TaMV infection or recovered from infection. These phenotypes were the result of a post-transcriptional gene silencing (PTGS) mechanism that targeted TaMV-CP sequences for degradation. The TaMV-CP sequences were degraded when present in the wild-type TaMV potyvirus, in transgene mRNA, or in chimeric viral vectors based on White clover mosaic virus. The more efficiently targeted region was mapped to a 134-nt segment. Differences were observed in the efficiency of targeting during cell-to-cell and long-distance movement of the chimeric viruses. However, the TaMV-CP sequences do not appear to be targeted for degradation when delivered by biolistics.

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Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA

Biochemical Journal ◽

10.1042/bj3630001 ◽

2002 ◽

Vol 363 (1) ◽

pp. 1-5 ◽

Cited By ~ 37

Author(s):

Malgorzata KISIELOW ◽

Sandra KLEINER ◽

Michiaki NAGASAWA ◽

Amir FAISAL ◽

Yoshikuni NAGAMINE

Keyword(s):

Adaptor Protein ◽

Human Cell Line ◽

Transcriptional Gene Silencing ◽

Specific Gene ◽

Post Transcriptional Gene Silencing ◽

Eukaryotic Genes ◽

Alternative Approach ◽

Genes Encoding ◽

The Common ◽

Interfering Rna

Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.

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