No abstract

We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or LlRn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (>400-bp) open reading frames, and a ca. 200-bp G+C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences.Repeated DNA sequences are present in the genomes of all metazoans (6). A class of highly repeated DNA that has been studied in primate and mouse genomes (1,10,14,18,19,31,32,37,49) has been called long interspersed repeated DNA (referred to as LINES [52] or, more recently, the Li family [59]). These families contain long members (several kilobases) that are repeated >20,000 times per genome and are responsible for the prominently stained electrophoretic bands that are seen when total genomic DNA is digested with the appropriate restriction endonuclease; the names of the endonucleases were originally used to denote these families.Extensive studies on mouse and primate LINEs (see references 43 and 53 for recent reviews) revealed several major features that these families share. First, many cloned members have, at what has been called the right or 3' end, a putative polyadenylation site, AATAAA, followed by an A-rich sequence (14,54,60). Second, although full-length members are 6 to 7 kilobases (kb) long (1, 14, 19), many cloned members are truncated and most often are missing a variable portion from their left end. Furthermore, there appear to be many more genomic copies of the right end than of the left end of certain cloned members of these families (14,19,59 by RNA polymerase 11 (20,50,56), although there are conflicting results about the extent to which transcripts are poly(A) + (26, 50) and to what extent LINE transcription may be asymmetric (50,56). It has been suggested that the truncated LINE members that end in A-rich 3' ends are incomplete DNA copies (retrotranscripts) of poly(A)+ LINE transcripts (14,43,55,59,60).The rat also contains a highly repeated family of transcribed LINE sequences (65). Members of this family have been (or still are) undergoing transposition in the rat genome, since at least three single-copy loci are polymorphic ow...

Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.

The large fragment of DNA polymerase I, isolated from Bacillus stearothermophilus, was used for dideoxy sequencing. This heat-stable enzyme permits performing sequencing reactions at high temperature to melt secondary structure and results in uniform band intensities and low background on the autoradiogram. The enzyme can be used in the standard Sanger one-step protocol or in a two-step protocol which separates the labeling reaction from the elongation-termination reaction. The enzyme can be used in double-stranded sequencing. 35S-labeled nucleotides may be used instead of 32P-labeled nucleotides. Both 7-deaza-dGTP and dITP can be used during the reaction in order to minimize band compression on the gel. Results presented here indicate that this enzyme should be a useful tool for sequence determination.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.