We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses ofthese sequences show (t) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical orign, (i) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (ii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. (ORFs). The HCV genome, however, displays singular characteristics at each terminus. We detect a hairpin structure at the 5' end of the genome as well as 5' and 3' subgenomic RNAs, the latter of which are polyadenylylated. These are consistent with a polyadenylylated 3' terminus of the viral genome and perhaps of functional subgenomic RNAs. Our data provide insights into the organization of the HCV genome, which may have important ramifications regarding the replication strategy and evolution of the virus. MATERIALS AND METHODSRNA was extracted from a high-titer plasma of an experimentally infected chimpanzee (6) and plasma (or serum) from HCV-positive or negative blood donors by a low-temperature guanidinium thiocyanate method (7). Poly(A)+ RNA was isolated from the liver ofthe same infected chimpanzee by the guanidinium thiocyanate/urea method (8). cDNA was synthesized from RNA according to Han and Rutter (9) and amplified by polymerase chain reaction (PCR) according to Saiki et al. (10). Briefly, RNA isolated from about 500 Al of plasma or S Ag of poly(A)+ liver RNA was converted into single-stranded cDNA by reverse transcriptase (BRL) using 150 pmol of the appropriate cDNA primer. For 5' end characterization by primer extension (7), first-strand cDNA was precipitated by spermine (11) and tailed with dA (9). Tailed or untailed cDNA was converted into double-stranded cDNA using a second-strand cDNA primer (9). This doublestranded cDNA was amplified using the indicated HCVspecific PCR primers for 35 cycles (940C, 1.5 min; 60'C, 2 min; 720C, 3 min). PCR without cDNA template was routinely performed to check for possible contamination during PCR. The PCR product was analyzed by Southern blot hybridization using a 32P-labeled oligonucleotide probe. The sequences and locations in the HCV genome of various cDNA and PCR primers are shown in Figs. 2 and 5. Most primers were designed to contain a Not I site for subsequent cloning of the PCR products into pUC18S, which contains a polylinker derived from Agt22 (9). DNA sequence was obtained by the supercoil sequencing (12) or the direct PCR sequencing method (13). RESULTSBased on the sequence of the major part of the HCV genome determined from overlapping A clones (2), we devised a directed strategy for obtaining clones representing the remaini...

Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 microM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO␥, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity.

Low-level B19 DNA was detected in nearly 1 percent of donations. The 23 percent of DNA-positive donations with both IgM and IgG B19 antibody most likely represent acute resolving infection, whereas those with IgG but no IgM are most consistent with a more chronic and possibly persistent phase of B19 infection.

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

We show that a family of prokaryotic repetitive sequences, called REP (repetitive extragenic palindromic), (Stern et al., 1984) is involved in the formation of chromosomal rearrangements such as duplications. The join‐points of seven RecA+ tandem duplications previously characterized in Salmonella typhimurium, that fuse the hisD gene to distant foreign promoters, were cloned and sequenced. In all seven cases they are shown to have originated by recombination between distant REP sequences. Importantly, several join‐points had also occurred at REP sequences even in a RecA‐background. Thus, REPs can recombine with each other by a RecA(‐)‐independent mechanism involved in the generation of chromosomal rearrangements. While all RecA+ duplications analysed resulted from recombination between REP sequences, some RecA‐duplications did occur also outside of REP sequences, in one case by recombination within a 7 bp homology. Possible roles for the known interaction between DNA gyrase and REP in chromosomal rearrangements are discussed.

WNV screening and supplemental assays had generally excellent analytical sensitivity, comparable to human immunodeficiency virus-1 and hepatitis C virus NAT assays. The presence of low-level viremic units during epidemic periods and the impact of MP dilutions on sensitivity, however, suggest the need for further improvements in sensitivity as well as a role for targeted individual-donation NAT in epidemic regions.

No abstract