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Cell-free DNA from dying cells has recently been discovered in human plasma. The authors investigated whether this cell-free DNA could cross the kidney barrier and be detected in urine and whether this could then be used as a diagnostic tool. Mice received subcutaneous injections of either human cells or purified human 32P-labeled DNA. The DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis and PCR. In humans, the ability of polymeric DNA to cross the kidney barrier was assessed by purification of DNA from urine and the subsequent detection of sequences that were different from human bulk nuclear DNA. Human-specific Alu sequences could be detected by PCR amplification of DNA purified from urine. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. Promega Wizard® Plus SV Minipreps Spin Columns(*) (see note below) were used for DNA isolation from urine.
Note: Wizard® Plus SV Miniprep Spin Columns are a component of the Wizard® Plus SV Minipreps DNA Purification Systems(*). See our online catalog for information on these specific systems.
Botezatu, I.1, Serdyuk, O.1, Potapova1, G.1, Shelepov, V.1, Alechina, R.1, Molyaka, Y.3, Ananev, V.2, Bazin, I.2, Garin, A.2, Navimanov, M.2, Knysh, V.2, Melkonyan, H.4, Umansky, S.4 and Lichtenstein, A.1 (2000) Clin. Chem. 46, 8.
1Institute of Carcinogenesis and 2Institute of Clinical Oncology, Cancer Research Center, Moscow, Russia 115478; 3Medical Genetic Center, Moscow, Russia 115478; 4DIAGEN Corporation, 6034 Monterey Ave., Richmond, CA 94805.
The posttranscriptional alterations of messenger RNA (mRNA) sequences, which can subsequently modify the information encoded by the RNA, occurs through a process termed RNA editing. In this paper, RNA editing in HIV-1 mRNAs in chronically infected H9 cells was investigated. The choice of an infected clonal cell line was critical to ensure that the observations of changes in mRNA sequences were posttranscriptional events and not emerging from variant provirus present in heterogeneous samples obtained from HIV-1-infected patients. The viral protein R (vpr) transcripts in H9 cells chronically infected with HIV-1 were isolated and amplified by RT-PCR using the PolyATtract® mRNA Isolation System (Cat.# Z5000 series) and the Access RT-PCR System(a) (Cat.# A1250), respectively, and the subsequent amplification products were cloned using the pGEM®-T Vector System(*) (Cat.# A3600). Mutations in vpr have been observed in T cells chronically infected with HIV-1, and thus the authors chose this region for investigation. The authors detected several base changes in the 5´-noncoding and vpr sequences in the viral mRNAs relative to the genomic sequence. Both G-to-A and C-to-U changes were observed. The RNA editing modifications observed were restricted to transcripts intended for processing and translation, suggesting that editing allows for a distinction between mRNA pools. The creation of stop codons in HIV-1 transcripts may control the translation of viral proteins, such as the viral R protein, that are involved in the regulation of HIV-1 expression and the survival of chronically infected cells. The authors also used the Beta-Actin Primer Pair(a) (Cat.# G5740) for these studies.
Bourara, K., Litvak, S. and Araya, A. (2000) Science 289, 1564.
Laboratoire de Replication et Expression des Genomes Eucaryotes et Retroviraux, UMR 5097, CNRS-Universite Victor Segalen-Bordeaux 2, and IFR 66 Pathologies Infectieuses, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France.
Nearly 40% of Familial Alzheimer's disease (FAD) cases result from mutations in one of the two human presenilin genes. Although the presenilins are conserved in multicellular organisms, from Arabidopsis thaliana to humans, their normal cellular functions are not well understood. To identify proteins that interact with Presenilin 1 (PS1), the authors screened for proteins that bound to PS1 from a human embryonic brain cDNA library using the yeast two-hybrid system. One clone encoding QM/Jif-1, a negative regulator of c-Jun, was isolated. Binding was confirmed using a pull-down assay and immunoprecipitation. Immunoelectromicroscopic analyses of brain tissue revealed that QM/Jif-1 colocalizes to the endoplasmic reticulum and the nuclear matrix. Chloramphenicol acetyltransferase assays in F9 cells indicated that wildtype PS1 suppresses transactivation by the c-Jun homodimer but does not affect transactivation by c-Jun/c-Fos heterodimers. In F9 cells wildtype PS1 suppressed retinoic acid-induced apoptosis, believed to be mediated in part by c-Jun. However, a PS1 construct containing a mutation that is associated with FAD was significantly less effective at suppressing retinoic acid-induced apoptosis. PS1 was also shown to promote the nuclear translocation of QM/Jif-1. These results are consistent with the suggestion that the Presenilins act as proteins responsible for intracellular transport and localization of a variety of cytoplasmic components and demonstrate that PS1 influences c-Jun activity and can inhibit apoptosis mediated through c-Jun pathways.
The authors amplified the full-length PS1 cDNA and subcloned it into the pCI-neo Mammalian Expression Vector(*) (Cat.# E1841). This construct was used as the template to create a construct containing a mutation associated with FAD. The pCI-neo Vector constructs were used in expression studies in F9 cells. The PS1 and various GST fusions protein were synthesized using the TNT® T7 and T3 Coupled Reticulocyte Lysate Systems(*) (Cat.# L4610, L4950). Apoptosis was assayed by the TUNEL method using Promega's Apoptosis Detection System, Fluorescein(*) (Cat.# G3250).
Imafuku, I.1, Masaki, T.2, Waragai, M.1, Takeuchi, S.1, Kawabata, M.3, Hirai, S.-i.4, Ohno, S.4, Nee, L.E.5, Lippa, C.F.6, Kanazawa, I.1, Imagawa, M.7 and Okazawa, H.1 (2000) J. Cell Biol. 147, 121.
1Department of Neurology, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan; 2The Third Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan; 3Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 170-0012, Japan; 4Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236, Japan; 5Family Studies Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892; 6Department of Neurology, MCP-Hahnemann University, Philadelphia, PA 19129; 7Laboratory of Environmental Biochemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Plant biologists have long suspected an evolutionary relationship between the green plants and red algae. Analyses of genomes and structures of chloroplasts (and mitochondria) to date have pointed to a single phyletic (monophyletic) origin of the green plants, red algae and glaucophytes, but such analyses of nuclear genes have been inconclusive. In this report an alternative nuclear marker--elongation factor-2 (EF-2)--was analyzed following RNA or genomic DNA isolation from multiple representative species. For the RNA isolation (from the euglenozoan Euglena gracilis), the researchers used the SV Total RNA Isolation System(*) (Cat.# Z3100) followed by RT-PCR to generate cDNA. Their findings support the hypothesis of an ancient endosymbiosis between a cyanobacterium and a eukaryote, which gave rise to kingdom Plantae. It is the first conclusive report of nuclear-encoded proteins (supported in addition by a similar analysis using a concatenate of 13 nuclear markers) being in agreement with the chloroplast and mitochondrial data. The authors state that the data shows a single primary endosymbiosis occurred prior to the evolutionary separation of the red algae and green plants.
Moreira, D.1,2, Le Guyader, H.1 and Philippe, H.1 (2000) Nature 405, 69.
1Equipe Phylogénie et Evolution Moléculaires, CNRS UPRES-A 8080, Université Paris-Sud, Bâtiment 444,91405 Orsay Cedex, France; 2 Departamento de Microbiología, Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain
Stem cell biology is rewriting our understanding of not so much how undifferentiated cells became differentiated, but how key signals can redirect the supposedly predetermined cell down an new path. For example, it's fairly well known that cell types can be redirected to generate a different cell type within the same germ layer but limited evidence for cell types being redirected to generate different cell types across germ layer. These researchers demonstrate how neural stem cells (NSCs), known to generate neurons, glia and blood cells, can be directed to generate muscle cells. NSCs are neuroectodermal in origin; mucsle cells are mesodermal. Primary and clonally derived adult NSCs were shown to generate skeletal muscle cells both in vitro and in vivo (following transplantation). Neural-derived muscle cells were detected by double immunofluorescence using Anti-b-Galactosidase mAb (Cat.# Z3781). SV Total RNA Isolation System (Cat.# Z3100) was used to isolate RNA from embryonic human NSCs (HENSCs)-injected and noninjected TA muscles, human myoblasts, undifferentiated/differentiated HENSCs. The Access RT-PCR System(a) (Cat.# A1250) was used to detect the presence of human MyoD transcripts in all RNA samples.
Galli, R.
1,2, Borello, U.2,3, Gritti, A.2, Minasi, M.G.3, Bjornson, C.4, Coletta, M.3, Mora, M.1, De Angelis, M.G.C.5, Fiocco, R.2, Cossu, G.2,4 and Vescovi, A.L.1,2 (2000) Nat. Neurosci. 3, 986.1National Neurological Institute, C. Besta, Via Celoria 11, Milan I-20133, Italy; 2Stem Research Intsitute, H.S. Raffaellee, Via Olgettina 58, Milan I-20133, Italy; 3Fondazione Pasteur-Cenci Bolognetti, Dept. of Histology and Medical Embryology, University of Rome, La Sapienza, Via A. Scarpa 14, Rome I-00161, Italy; 4Dept. of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195-7350 USA; 5Department of Experimental Medicine, University of Pavia, Via Forlanini 8, Pavia I-27100, Italy.
Pre-eclampsia is a major concern during first pregnancies regardless of race and culture. Serious pre-eclampsia can lead to death. Though the etiology and pathogenesis have yet to be elucidated, it is assumed that the placenta is involved, as termination of pregnancy results in recovery from this disease.
These researchers cloned a cDNA from human placental mRNA that coded for a 121-amino acid precursor protein. The mature protein was found to be identical to neurokinin B (NKB). (In pregnant rats, NKB possesses 'pressor' activity.) In humans, elevated NKB is seen in pregnancy-induced hypertension as well as pre-eclampsia. Also, NKB is localized to the outer syncytiotrophoblast in humans, the region of the placenta known to be involved in pre-eclampsia. When questioning whether neurokinin B could be involved in the pathogenesis of pre-eclampsia, the authors noted that NKB could not cause the failed trophoblast invasion but that its potent vasoactive properties might be involved in the clinical manifestations of pre-eclampsia.
Human placental RNA was obtained from pregnancies terminated at 9 and 13 weeks, as well as at term, in compliance with and approval from the Local Research Ethics Committee. RNA was extracted and the full-length preproNKB precursor amplified from total RNA. 3´ RACE was performed using a 5´ gene-specific primer derived from the human cDNA clone 138761. The PCR fragment was purified following gel electrophoresis and cloned into the pGEM®-T Easy Vector System(*) (Cat.# A1360), sequenced and compared with the GenBank® database.
Page, N.M.1, Woods, R.J.1, Gardiner, S.M.2, Lomthaisong, K.1, Gladwell, R.T.1, Butlin, D.J.1, Manyonda, I.T.3 and Lowry, P.J.1 (2000) Nature 405, 797.
1School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK; 2School of Biomedical Sciences, The University of Nottingham, Nottingham NG7 2UH, UK; 3Department of Obstetrics and Gynaecology, St George's Hospital, London SW17 0QT, UK.