No abstract

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNAdependent RNA polymerase. By mimickisg the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1-to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.The transfer of genetic information from RNA to DNA and then back to RNA is a scheme characteristic of retroviruses. Such a scheme provides a mechanism for the replication of RNA genomes (reviewed in refs. 1-3). In exploring variations of an in vitro transcription-based amplification system (TAS) (4), it was discovered that it was possible to devise a concerted, three-enzyme, in vitro reaction to carry out an isothermal replication of target nucleic acid sequences, analogous to the strategy used in retroviral replication. This reaction is a self-sustained sequence replication (3SR) system involving the collective activities of avian myeloblastosis virus (AMV) reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase. The accumulation ofboth target nucleic acid-specific RNA Approximately 25 mg (wet weight) of Trisacryl beads containing oligonucleotide 86-273 (5'-AGTCTAGCAGAA-GAAGAGGTAGTAATTAGA-3') was prehybridized in 250 pA of hybridization solution (5 x standard saline phosphate/ EDTA/10% dextran sulfate/0.1% SDS) in a 2-ml microcolumn (Isolab) for 30 min at 370C. The prehybridization solution was removed, and 40 p.1 of fresh hybridization solution was added to the beads, together with the 60 p.1 of solution from the solution hybridization step. The beads were then incubated at 370C for 1 hr with occasional mixing and then washed six times with 1 ml of 2x standard saline citrate (6) at 370C. The radioactivity of the beads and the combined washes was measured by Cerenkov counting for 1 min. The amount of target detected was determined by calculating the percentage of total radioactivity captured on the beads and Abbreviations: 3SR, self-sustained sequence replication; TAS, transcription-based amplification system; HIV-1, human immunodeficiency virus type 1; AMV, avian myeloblastosis virus.§To whom reprint requests should be addressed. 1874The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci...

Phospholipid (PL) scramblase is a 35 kDa protein that is thought to mediate Ca2+-induced bidirectional transbilayer movement of plasma membrane phospholipids in activated, injured, or apoptotic cells. We recently reported the molecular cloning of a PL scramblase of human (HuPLSCR1) and mouse origin, respectively. In the present study, the gene for HuPLSCR1 was cloned from a human genomic library. The gene size is 29.7 kb and includes nine exons. Analysis of the 5' flanking genomic sequence with luciferase reporter constructs located the promoter to a region spanning from -95 to +60 of the first (untranslated) exon. Furthermore, we report the molecular cloning of three additional novel cDNAs encoding proteins with high homology to HuPLSCR1. The predicted open reading frames encode proteins with 59% (HuPLSCR2; 224 aa), 47% (HuPLSCR3; 295 aa) and 46% (HuPLSCR4; 329 aa) identity, respectively, to HuPLSCR1. All members of the PLSCR gene family conserve those residues contained in the segment of the PLSCR1 polypeptide that was previously shown to bind Ca2+. With the exception of HuPLSCR2, these proteins also each contain multiple PXXP motifs and a PPXY motif located near the N-terminus, implying the potential for interaction with SH3 or WW domain-containing proteins, respectively. HuPLSCR1, 2, and 4 were found to be closely clustered on chromosome 3 (3q23), whereas HuPLSCR3 is located on chromosome 17. Northern blots revealed that the expression of HuPLSCR2 is restricted to testis, whereas HuPLSCR1, 3 and 4 are expressed in most of the 16 tissues examined. Notable exceptions were HuPLSCR4, which was not detected in peripheral blood lymphocytes, and HuPLSCR1 and HuPLSCR3, which were not detected in brain.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in approximately 100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300-400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver approximately 10- to 50-fold as compared with PL alone.

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.