Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni.

We have isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage may of five of the clones spanning 35 kilobase pair (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5' end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotides. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed several very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene.

Egg production by worm pairs is a major cause of pathogenesis in schistosomiasis. To further the understanding of female reproductive development, we have isolated and characterized a complete copy of an eggshell protein precursor gene, p48. Sequence analysis reveals that the gene has 3 open reading frames and does not contain an intron. One of the open reading frames, ORF1, encodes a polypeptide of 50 kDa which shows strong homology to insect chorion proteins. Determination of the position of the mRNA cap-site facilitated identification of putative regulatory elements in the 5' upstream region of the gene. Some of these elements (e.g., TCACGT) have been shown to play a role in the regulation of chorion gene expression in insects. p48 mRNA is detectable only in mature female worms and the ability to detect the mRNA coincides temporally with worm pairing. Quantitative comparisons, during female reproductive development, of p48 transcripts to those from another eggshell protein precursor gene, p14, show that the p48 mRNA is significantly less abundant than p14 mRNA. In mature female worms, p48 mRNA can only be detected in vitelline cells. Antibodies made against the polypeptide sequence deduced from ORF1 of the p48 gene recognize a 50-kDa molecule in extracts from mature female worms, but not in extracts from immature females or males.

Organization of the ribosomal RNA genes in Schistosoma mansoni.

The organization of the rRNA genes of Schistosoma mansoni has been determined by Southern blot analysis of genomic DNA digested with restriction enzymes, by isolation of the entire repeat on a single fragment of about 11 kilobase pairs from a genomic DNA library constructed in bacteriophage lambda and by characterization of three cloned EcoRI fragments which span the entire repeat. The segments encoding both the large and small rRNA subunits have been identified using specific cloned yeast rDNA fragments as probes and EcoRI, HindIII and BamHI restriction enzyme maps of the rRNA genes were constructed. The ends of the RNAs have been precisely mapped on the genomic DNA by S1 protection experiments. Our data indicate that the rRNA genes are present as a tight cluster. The total length of the rDNA repeat is about 10 kilobase pairs. There appears to be no variation in the size of transcribed and non-transcribed spacer DNA. At the RNA level we have characterized and mapped a small gap in the 28S RNA molecule. The interruption causes the RNA to dissociate into two equal sized fragments when analyzed under denaturing conditions.

A cloned DNA probe identifies the sex of Schistosoma mansoni cercariae.

This report describes a method for the identification of the sex of Schistosoma mansoni cercariae using a cloned DNA probe which is female-specific. The probe is a 339 bp repeat; the sequence is presented. Cercariae from nine mono-miracidially infected snails were used in a double blind study. Mice were infected and the sex of adult worms observed. DNA was isolated from cercariae, digested with EcoRI, subjected to agarose gel electrophoresis, transferred to a matrix and hybridized with the cloned female-specific DNA probe. In all nine cases the sex of the cercariae as determined by DNA analysis agreed with the sex of the adult parasites.

Characterization of a 54-nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni.

We have analyzed 572 bp in the 28S rDNA of the human blood fluke Schistosoma mansoni which correspond to expansion segment 5 of domain IV as defined by Clark et al. for the Xenopus laevis 28S rRNA. S1 nuclease mapping and primer extension analysis comparing this region with the mature 28S rRNA indicate that there are 54 nucleotides present in the 28S rDNA which are absent from the mature rRNA. This defines a gap that creates two 28S rRNA subunits (28S alpha and 28S beta). Comparison of the S. mansoni sequence with rDNAs of other organisms which contain gaps in their 28S rRNA shows that the overall features are conserved except that the S. mansoni gap is less A + T-rich. The conserved features include: (1) the location of the gap within the 28S rRNA; (2) the predicted secondary structure of the gap, containing a stem-loop with a UAAU sequence within the loop; and (3) a conserved CGAAAGGG on the 3' side of the gap.

Schistosoma mansoni tropomyosin: production and purification of the recombinant protein and studies on its immunodiagnostic potential.

A cDNA that encodes Schistosoma mansoni tropomyosin, except for 10 amino acids at the amino terminus, has been cloned into a pOTSNCO plasmid vector. Induced expression resulted in a constant level of recombinant protein production. The recombinant S. mansoni tropomyosin was purified from preparative SDS-PAGE gel and by a combination of 20% ammonium sulfate fractionation and fast protein liquid chromatography-ion-exchange chromatography. The purified recombinant S. mansoni tropomyosin was tested as an immunodiagnostic reagent in Western blot and enzyme-linked immunosorbent assays. Sera from individual patients with chronic S. mansoni infection, but not S. haematobium, S. japonicum, parasitic infections other than schistosomiasis, and without infection reacted with the recombinant tropomyosin. The species specificity of S. mansoni tropomyosin suggests that further study of its potential as an immunodiagnostic reagent is warranted.

Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.

Schistosoma mansoni: identification and characterization of schistosomula polypeptides.

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.

Schistosoma haematobium: analysis of eggshell protein genes and their expression.

Eggshell protein genes of Schistosoma mansoni that encode a 14 kDa protein have been shown to be highly conserved and expressed in a sex-, tissue-, and temporal-specific manner. To initiate studies on the eggshell protein genes of S. haematobium, a cDNA probe, pSMf 61-46, representing a S. mansoni eggshell protein mRNA was used to screen a S. haematobium genomic library. Of the seven independent recombinant clones isolated, two (lambda SH 2-1 and lambda SH 6-1) were analyzed and compared to those of S. mansoni. lambda SH 2-1 and lambda SH 6-1 each contain a different genomic copy of the gene encoding a 19.8 and 17.6 kDa protein, respectively. This is due to an additional 78 bp present in the coding region of lambda SH 2-1 relative to lambda SH 6-1. The rest of the coding sequences are identical, and the 5' and 3' untranslated regions are nearly identical. The deduced amino acid sequences of S. haematobium eggshell proteins are very rich in glycine (47 and 50%) when compared to 43.5% glycine in the protein encoded by S. mansoni. Long stretches of glycines, as many as 15 in a row, occur in the S. haematobium sequence. DNA comparison of the eggshell protein genes of the two schistosome species yielded an overall homology of 83.1%. The homology is much higher in the 5' and 3' untranslated regions than in the protein-coding regions. Genomic clones of both species contained second open reading frames, which appeared to be kept open as a consequence of the amino acid composition of the other. There are no introns in S. haematobium or S. mansoni eggshell protein genes, and the genomic Southern data indicated a similar arrangement of these genes in the genome of both species. Primer extension experiments and dideoxynucleotide sequencing of the RNA determined the mRNA cap site sequence as ATCAT and ATCAC in lambda SH 2-1 and lambda SH 6-1, respectively. Northern blot analysis determined the size of the mRNA to be about 1.0 kp. Expression of the RNA from these genes appears to be regulated in a manner similar to the corresponding genes in S. mansoni. mRNA is found only in mature females and first appears at 70 days after infection of hamsters. DNA sequence comparisons of the 5' flanking regions of S. haematobium and S. mansoni eggshell protein genes to each other and to those of silkmoth and Drosophila revealed several short sequence elements that are shared.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

Catalysis of adenovirus DNA synthesis in vitro by DNA polymerase gamma.

DNA replication complexes were purified from adenovirus type 5 (Ad5)-infected HeLa cells. DNA synthesis by these complexes in vitro was extremely sensitive to the inhibitors dideoxythymidine triphosphate, N-ethyl maleimide and p-hydroxymercuribenzoate. The bound DNA polymerase was released from the complexes by limited digestion with micrococcal nuclease. This released polymerase preferred poly(rA):(dT)12-18 as template over activated calf thymus DNA. These results are compatible with the major polymerase in the replication complex being of the gamma class.

Characterization of two temperature-sensitive mutants of adenovirus type 5.

The properties of two temperature-sensitive mutants ts 18 and ts 19 of adenovirus type 5 were studied. It was demonstrated that they had a defect such that they failed to assemble virus and showed defective processing of infected cell polypeptides at the restrictive temperature. Analysis, after protease digestion, of the virions produced at the permissive temperature by SDS PAGE, and of the substrate availability of the mutants to the virus protein kinase suggested that polypeptide VI was defective in these mutants.

Schistosoma mansoni: identification and analysis of an mRNA and a gene encoding superoxide dismutase (Cu/Zn).

The sequence of an mRNA encoding superoxide dismutase (EC 1.15.1.1) of Schistosoma mansoni has been determined. The deduced amino acid sequence which it encodes shows 40 to 45% homology with the amino acid sequences of the cytosolic superoxide dismutase from 10 other species, as well as similar homology with the human extracellular form. Hybridization selection of the mRNA by cDNA clone pSM38 and in vitro translation of this mRNA have consistently produced a protein of 20,000 Da and occasionally, a 40,000-Da protein. Both are immunoprecipitable with sera pooled from patients with chronic schistosomiasis. The gene from which this mRNA is transcribed has also been isolated and characterized. It spans 5.1 kb of chromosomal DNA and possesses three exons and two introns which interrupt the coding region. These introns contain the splice junction sequences which fit the consensus sequences observed in mammalian genes. The upstream sequence of the gene shows a region for a potential 'TATA' box and the downstream sequence contains two polyadenylation sites.

Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni.

We have isolated and characterized a cDNA clone that is derived from a developmentally regulated mRNA found only in mature female schistosomes. The mRNA is approximately 950 nucleotides in length and is not detectable in immature female schistosomes isolated from single-sex infections, in male worms, or in eggs. During normal bisexual infections, the mRNA species is first detected 28 days after infection (the time of worm pairing) and increases to a high level 35 days after infection, coinciding with the start of egg production. The nucleotide sequence of the cDNA shows two large open reading frames in the coding strand. Several features of the clone, including the deduced sequence of the polypeptide encoded by one of the reading frames, suggest a relationship to the silk moth chorion (egg shell) gene family. The isolation of this clone provides us with a probe for further studies of female schistosome development and is a first step toward a detailed understanding of this process at the molecular level.

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens.

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.

A retroposon-like short repetitive DNA element in the genome of the human blood fluke, Schistosoma mansoni.

The genome of Schistosoma mansoni, a human blood fluke, contains a family of short repetitive DNA elements which we have named the SM alpha family. In this paper we report the sequences of two SM alpha family members which are derived from tandem arrangements and four family members which are dispersed copies. The two tandemly repeated copies are 331 and 335 bp, while the four dispersed copies range in size from 107 to 322 bp. Three dispersed copies are flanked by direct repeats and have AT-rich 3' ends. The tandem copies and one of the dispersed copies have regions of homology to RNA polymerase III promoters and arginine tRNA genes. In addition the repeated element is rearranged in two of the dispersed copies when compared with the other dispersed and two tandem copies. Localization studies show that SM alpha elements are distributed in the sex and autosomal chromosomes. These observations suggest that members of this family may have been dispersed throughout the genome via RNA intermediates.

Schistosoma haematobium and S. japonicum: analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences.

We have determined the intragenic organization of the rRNA genes of Schistosoma haematobium and S. japonicum and found them to be similar to that of S. mansoni and other eukaryotes. An entire ribosomal repeat approximately 10 kbp in size from each species was isolated as a SalI fragment from a genomic library constructed in bacteriophage lambda. The segments encoding both the small and large rRNAs have been identified using three cloned EcoRI fragments of S. mansoni as probes. There were three EcoRI fragments (4.2, 3.0, 1.6 kbp) from S. haematobium and four EcoRI fragments (4.6, 2.3, 1.7, 1.0 kbp) from S. japonicum. As in a wide variety of organisms within the protostome phyla, the 28S rRNA in schistosomes contains a "gap" which separates it into two fragments. The length of the gap sequence in S. haematobium is 54 bases and it is identical to that in S. mansoni in both length and sequence. However, in S. japonicum the sequence is between 64-67 bases long. In each case, irrespective of the species, the gap is located at the same position within the 28S rRNA. Secondary structures of the gap sequence derived by computer analysis predict a conformation with the minimum free energy that has an UAAU tract in a hairpin loop for S. haematobium and an UAUU tract for S. japonicum.

Design and use of an inducibly activated human immunodeficiency virus type 1 Nef to study immune modulation.

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell surface proteins, and associate with many intracellular proteins that are thought to facilitate HIV infection. One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells. This has been attributed to effects of Nef on cell proliferation and apoptosis. We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated. It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steric hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef-ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (lipid rafts) only after activation. Using this inducible Nef, we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line. Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT. These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef. The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion.