The AMPLICOR® HCV Tests for the Detection and Quantitation of Serum or Plasma HCV RNA.

Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.

Transcription of the HIV-1 LTR is regulated by the density of DNA CpG methylation.

Transcription from the HIV-1 long terminal repeat (LTR) was shown to be inhibited by DNA CpG methylation both in vivo and in vitro. Enzymatic methylation of CpG sites localized within the LTR decreased the transcription of the CAT reporter gene, chloramphenicol acetyltransferase, as assayed by the transient expression of this gene in tissue culture. The inhibitory effect could be initially overcome, in trans, by the transactivator tat. As a function of time, the presence of tat had no observable effect on transcription, within the limits of detection sensitivity, suggesting that the level of basal transcription was reduced to very low levels. This effect is suggestive of the involvement of cellular CpG methylation-dependent inhibitory factors which have been characterized by other laboratories. These data imply that transactivation is reduced to low levels after longer periods of time when the DNA template is sparsely methylated. The transcriptional inhibitory process may involve proteins such as MeCP which may interact with methylated DNA more slowly and/or weakly. Conversely, densely methylated DNA was transcriptionally repressed immediately which suggests the rapid/strong association of the cellular inhibitory factor(s). The transcriptional inhibitory effect was also observed in an in vitro transcription run-off system. These data suggest that the methylation-mediated inhibition of transcription is directly affected by CpG methylation density and may involve other factors.

A filamentous-like mutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellular protein invades and grows in 3T6 and Caco-2 cells.

We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.

Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19).

Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.

Bovine lens membrane proteins: MP70, MP64, and MP38 are products of the same gene.

We have carried out limited microsequence analysis of bovine lens intrinsic membrane proteins having molecular weights of 70, 64, and 38 kD. These three polypeptides all have an identical amino acid terminal sequence, at least for the first 17 amino acid residues, indicating a common origin. When calf lens RNA was hybridized with a labeled antisense oligonucleotide common to the amino acid sequence of these three polypeptides, a single message with an apparent molecular size of 2.6 kb was detected. Together, these results indicate that bovine lens MP70, MP64, and MP38 are products of the same gene and that the lower molecular weight polypeptides are the result of degradation (processing) of lens MP70 at its COOH-terminal end.

Bovine lens 23, 21 and 19 kDa intrinsic membrane proteins have an identical amino-terminal amino acid sequence.

We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino-terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.