A shortcut in phage screening technique

Andrade, Alexandre de Melo; Rezende‐Teixeira, Paula; Siviero, Fábio; Santelli, Roberto Vicente

doi:10.1590/s1415-47572005000100025

Cited by 3 publications

(3 citation statements)

References 2 publications

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“…The protocol followed was a modification of CsCl lambda DNA purification described in Santelli and Navarro-Cattapan (2000) and in Andrade et al (2005).…”

Section: Rhynchosciara Americana Genomic Phage Library Screeningmentioning

confidence: 99%

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

et al. 2009

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An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

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“…The protocol followed was a modification of CsCl lambda DNA purification described in Santelli and Navarro-Cattapan (2000) and in Andrade et al (2005).…”

Section: Rhynchosciara Americana Genomic Phage Library Screeningmentioning

confidence: 99%

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

et al. 2009

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“…The treatment of membranes and hybridizations were as described in Sambrook and Russell (2001). After the first screening, the protocol followed to isolate the positive recombinant was as described in Andrade et al (2005) and the lambda DNA purification was done followed the protocol described in Santelli and Navarro-Cattapan (2000).…”

Section: Library Screeningmentioning

confidence: 99%

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

et al. 2009

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Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5'-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

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“…Washes were done four times at 65-C with 2Â SSC, 0.1% SDS and twice with 0.1Â SSC, 0.1% SDS and exposed to a PhosphorImager system (Storm, Molecular Dynamics). After the first screening, the protocol for isolation of the positive recombinant was as described by Andrade et al (2005) and the lambda DNA purification was done following the protocol described by Santelli & Navarro-Cattapan (2000).…”

Section: Library Screeningmentioning

confidence: 99%

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

et al. 2008

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Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

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A shortcut in phage screening technique

Cited by 3 publications

References 2 publications

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

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