Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method.

A solid phase assay for the colorimetric detection of competitively amplified HCV-cDNA has been established and used to investigate clinical samples from patients with chronic hepatitis. The assay is based on the reduction in the amplification of an hepatitis C virus-related competitor molecule by wild-type hepatitis C virus during polymerase chain reaction. The internal standard contains a lac operator sequence, allowing the amount of amplified competitor to be determined using a lac I-repressor/beta-galactosidase fusion protein. The reduction in the amplification of competitor is dependent upon the concentration of HCV-RNA in the original sample. External hepatitis C virus wild-type standards are used to calibrate each concurrently tested set of patients. We present and discuss the potential benefit, but also the limitations of this new approach for quantifying hepatitis C virus viremia. In 47 serum samples from 28 patients with chronic hepatitis C virus infection, including five repeatedly tested alpha HCV positive patients under interferon therapy, viral titer was determined. Sera from nine healthy blood donors served as controls. The sensitivity and specificity of this procedure are identical to those of conventional nested polymerase chain reaction. As both internal and external standards are used in every assay and final detection of amplicons can be carried out in microtiter plates, this reliable and time-saving test system may be routinely applied for monitoring antiviral treatment or for studying the relation of plus- and minus-stranded HCV-RNA in infected tissues.

Characterization of the complete protein disulfide isomerase gene of Schistosoma mansoni and identification of the tissues of its expression.

cDNA and genomic clones of Schistosoma mansoni containing the complete sequence of a homolog of protein disulfide isomerase have been identified. The protein disulfide isomerase gene in schistosomes is a single copy gene having a genomic structure that is very similar to that of man. The C-terminus of the deduced protein is KDEL which in mammals functions as a signal sequence for retention of luminal proteins in the endoplasmic reticulum. Immunohistology and in situ hybridization identify the gastrodermis of the gut, the wall cells of the protonephridia, and the sustentacular cells of the testes to be the major tissues of protein disulfide isomerase gene expression. The protein disulfide isomerase of schistosomes, produced in an expression vector in Escherichia coli, catalyzes disulfide/sulfhydryl isomerization in vitro.

Ferritins of Schistosoma mansoni: sequence comparison and expression in female and male worms.

Recombinant clones of Schistosoma mansoni cDNA libraries containing the complete coding regions of 2 different ferritin subunits have been isolated and sequenced. This allows for the first time a comparison of ferritin sequences from an invertebrate with those of vertebrates. The deduced amino acid sequences of both Schistosoma ferritin subunit clones show significant homology to vertebrate ferritin H chains. Similarity exceeds 50% identity and includes the recently identified ferroxidase center which is present only in H chains. However, non-conservative substitutions of amino acid residues lining the 3-fold symmetry channel were found, and a gap of 3 successive amino acids unique to the 2 Schistosoma ferritin sequences was identified. Remarkably, for each of the 2 genes, we found a conspicuous difference in the amount of ferritin transcripts between females and males: one of the genes is preferentially expressed in females, the other in males.

Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.

Butyrate effect on nuclear proteins of two Chinese hamster cell lines.

The effect of sodium butyrate on the nuclear proteins of two Chinese hamster cell lines (V79 and CHO) was studied. Butyrate treatment induces hyperacetylation of core histones in both cell lines, while H1 histone shows a different behavior. In CHO cells H1 is dephosphorylated following butyrate incubation; V79 do not show any change of H1 subtypes. It seems that H1 response to butyrate treatment is cell type dependent. Using silver staining a group of proteins that could be present in vivo in the nucleo-protein complex was also detected.

Gender-specifically expressed genes in Schistosoma mansoni.

Using a genomic gene bank in phage lambda and two cDNA banks in the expression vector lambda-gt11 we have cloned and characterized genes that are expressed preferentially or exclusively in females. One of these genes transcribes two predominant RNA molecules of 0.8 and 3.9 kb which comprise more than 5% of the mRNA population of adult female worms. Transcription of these two RNAs occurs in close proximity on the genome, probably in an overlapping fashion. Experiments are presently in progress to sequence the genes and to produce antibodies against their polypeptide products which will be used to determine in which tissue and at what time in development these genes are expressed. The gene products are probably used for egg shell formation. The final long-term perspective of this project is to interfere with the schistosome parasite's cycle and to reduce its pathogenicity by interrupting egg production.

Nonpackaging and packaging proteins of hnRNA in Drosophila melanogaster.

Monoclonal antibodies have previously been raised against chromosomal proteins of Drosophila. Using a biochemical fractionation method for the isolation of large hnRNA-containing structures (hnRNP) of Drosophila tissue culture cells, we show that seven of these antibodies recognize different antigens, and that these antigens are associated with RNA. Analysis of the sedimentation behavior of antigen-containing structures in sucrose gradients reveals that the antigens are differentially distributed with respect both to one another and to pulse-labeled RNA. We demonstrate that the antigens are minor components of hnRNP and are different from the major Drosophila hnRNP packaging proteins, which we have also identified. The antigens are probably involved in the processing of hnRNA in the nucleus.

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster: establishment of antibody producing cell lines and partial characterization of corresponding antigens.

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.

Initiation of lambda DNA replication in vitro promoted by isolated P-gene product.

The product of the P gene of bacteriophage lambda was isolated from heat-induced lambda-lysogenic Escherichia coli cells. It was found to bind to DNA, to be devoid of nuclease activity acting on double-stranded lambda DNA and of nicking/closing activity. Initiation of lambda DNA replication promoted by the P-gene product in a complementation assay in vitro was sensitive to rifampicin. Sedimentation analysis of the products and their hybridization to separated lambda DNA strands indicate that lambda DNA was formed in a reaction similar to ring-to-ring replication in vivo. The reaction was symmetric from the beginning, i.e. both lambda DNA strands were copied without delay.