Nucleotide sequence analysis of the inversion termini located within IS3 elements α3β3 and β5α5 of Escherichia coli K-12

This paper presents the first detailed structural analysis of termini of an inversion mediated by recombination between Escherichia coli native IS elements. The complete nucleotide sequence of the inversion termini in the lactose region of Escherichia coli K-12 confirms our previous suggestion that the inversion occurred by homologous recombination between α3β3 and β5α5 IS3 elements (D.J. Savic, J. Bacteriol. 140:311-319, 1979; D.J. Savic, S. Romac, and S.D. Ehrlich, J. Bacteriol. 155:943-946, 1983). The data show a slight structural divergence of α3β3 and β5α5 elements, but they do not reveal new sequences within recomhined IS3 elements that could influence the expression of nearby genes

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

<p>Abstract</p> <p>Background</p> <p>Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by <it>ERBB2</it> (<it>HER-2/neu</it>) oncogene expression.</p> <p>Results</p> <p>The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of <it>ERBB2</it>-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of <it>ERBB2</it>. The relative expression balance between AS variants from 3 genes was differentially modulated by <it>ERBB2</it> in this model system.</p> <p>Conclusions</p> <p>In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by <it>ERBB2</it>-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.</p

Transcriptomic Analysis of Human Retinal Detachment Reveals Both Inflammatory Response and Photoreceptor Death

Background Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. Methodology/Principal Findings Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. Conclusions/Significance This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery

Transcriptional analysis of the bovine herpesvirus 1 Cooper isolate

Blot hybridization analysis of infected bovine herpesvirus 1 (BHV-1) cellular RNA isolated at various times post infection and after treatment with specific metabolic inhibitors was used to characterize transcription of the BHV-1 Cooper isolate. Synthesis of BHV-1 RNA was detected as early as 3 h post infection and reached a maximum at six to eight hours post infection. The most transcriptionally active area of the genome was between map units 0.110 to 0.195, within the Hin dIII I fragment. From the entire genome a total of 59 transcripts ranging in size from approximately 0.6 to 10 kilobases were characterized as belonging to one of three distinct classes. Using the protein synthesis inhibitor cycloheximide, three immediate-early transcripts were identified as originating from the internal inverted repeat region between map units 0.734 and 0.842, corresponding to the Hin dIII D fragment. Using phosphonoacetic acid to prevent virus DNA synthesis by inhibition of the BHV-1 DNA polymerase, 28 early transcripts were recognized. The remaining 28 transcripts, classified as late RNA, were detected without the use of metabolic inhibitors at 6 to 8 h post infection. Transcription of early and late RNA was not restricted to any specific area of the genome. Eighty percent of the transcripts from both the Hin dIII A fragment, between map units 0.381 to 0.537 within the unique long segment, and the Hin dIII K fragment, between map units 0.840 to 0.907 of the unique short segment, were designated as belonging to the early class.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41672/1/705_2005_Article_BF01316744.pd

Hybrid P(L)tl promoter with dual regulation control

The newly constructed P(L)tl hybrid promoter is composed of the operator and promoter sequences of tac and the -35 to -135 region of the phage λ P(L) promoter, which contains the AT-rich block and the OL2 and OL3 segments. Transcriptional properties of P(L)tl were examined and compared with tac and λ P(L) as reference promoters. The hybrid P(L)tl exhibits different and improved properties over tac promoter in four ways: (i) when repressed, the repression is almost complete; (ii) after complete induction, the hybrid P(L)tl promoter shows a 1.4-2 times higher expression; (iii) the hybrid P(L)tl promoter permits flexible gene expression because it can be utilized under either or both repression controls simultaneously; (iv) the P(L)tl promoter permits enhanced expression of genes encoding products with unknown properties. When compared with the strong promoter P(L) from phage λ, results with the P(L)tl promoter in lacZ fusion constructs show higher levels of β-galactosidase activity. We also constructed a plasmid vector, pP(L)tl7G, which contains the hybrid P(L)tl promoter with a polylinker sequence at its 3' end, which facilitates efficient fusions of foreign genes in any of the reading frame

Constitutive interferon expression from retroviral vector

A genomic fragment with the human β-interferon gene was cloned into a pL3-4, a defective Moloney murine leukemia virus (M-MuLV) vector. Here we show that clones selected after viral infection of mouse NIH 3T3 cells constitutively produced 128 IU/ml of human β-interferon. Constitutive synthesis of retroviral RNA was confirmed by dot blot hybridization of RNA isolated from two of the selected clones. Poly(I)·poly(C) and cycloheximide induction resulted in an increased RNA level, but this was not reflected in an increased production of biologically active interferon