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Successful vaccination of mice against an otherwise lethal challenge with the Peru strain of Trypanosoma cruzi necessitates the induction of a strong cell mediated immune response. Previously, immunization of mice with the paraflagellar rod proteins from Trypanosoma cruzi90% reduction in parasitemia in immunized mice challenged with the bloodstream stage of Trypanosoma cruzi.

The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa. Based on these similarities, we have designated this gene family tolT. Immunization of mice with recombinant TolT generates a population of CD4(+) T lymphocytes that recognize T. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-gamma), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-gamma and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected with T. cruzi also produce significant levels of IFN-gamma when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T. cruzi.

Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50-90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putative N-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner. For the first time it was possible to assign a defined ligand to a sequenced glycoprotein belonging to the gp85 family. This fact, together with the reported binding of family members to cell surfaces, reinforces the hypothesis that this family encodes glycoproteins with similar sequences but differing enough as to bind to different ligands and thus forming a family of adhesion glycoproteins enabling the parasite to overcome the barriers interposed by cell membranes, extracellular matrices, and basal laminae.

The major structural proteins present in the paraflagellar rod of Trypanosoma cruzi migrate on SDS-polyacrylamide gels as two distinct electrophoretic bands. The gene encoding a protein present in the faster migrating band, designated PAR 2, has been identified previously. Here we report the isolation and partial characterization of three genes, designated par 1, par 3, and par 4, that encode proteins present in the two paraflagellar rod protein bands. Peptide-specific polyclonal antibodies and monoclonal antibodies against the four proteins encoded by these genes shows that PAR 1 and PAR 3 are present only in the slower migrating paraflagellar rod band, and that PAR 2 and PAR 4 are present only in the faster migrating band. Analysis of the nucleotide sequence of these genes and the amino acid sequence of the conceptual proteins encoded by them indicates that par 2 shares high sequence similarity with par 3 and both are members of a common gene family, of which par 1 may be a distant member. Analysis of gene copy number and steady-state RNA levels suggest that the close stoichiometric ratio of the four PAR proteins is likely maintained by homeostatic regulation of RNA levels rather than gene dosage.

Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.

We have examined the ability of mice to survive a lethal challenge with the parasitic hemoflagellate, Trypanosoma cruzi, following immunization with paraflagellar rod proteins (PAR) 1 and 2 either alone or in combination with the following adjuvants: Freund's, alum, QS-21, Ribi-700, or IL-12. PAR administered subcutaneously (sc) in combination with Freund's or alum provided significant protection, 100 and 83%, respectively, against a T. cruzi challenge. In contrast, PAR in combination with QS-21, Ribi-700, IL-12, or Freund's administered intraperitoneally (ip) or PAR alone provide no protection against a challenge. PAR-specific serum antibody titers and isotype profiles for several of the immunization regimens were determined, and no positive correlation could be seen between a protective immune response and either antibody titer or the subclass of antibody induced. We also examined the ability of PAR to stimulate T cells from the spleen and lymph nodes of mice immunized with PAR in combination with Freund's (sc), Freund's (ip), alum, or Ribi-700. Each of the adjuvants strongly enhanced the ability of enriched T cells to proliferate in a PAR-specific fashion, suggesting no obvious correlation between PAR-specific T cell activation and protection. However, examination of the cytokine profiles of the stimulated T cell groups showed that the protective groups differed from the nonprotective groups. While all four groups showed low levels of IL-10, the Freund's (sc) and alum groups had higher levels of IFN-gamma and IL-2 than Freund's (ip) and Ribi-700 groups, and most strikingly, no IL-4 could be detected in either the Freund's (sc) or the alum group, in contrast to significant levels of IL-4 in both the Freund's (ip) and the Ribi-700 group. These findings indicate that protective immunity in mice immunized with PAR is associated with a Th1-type response.

The paraflagellar rod proteins (PAR) purified from Trypanosoma cruzi epimastigotes were shown to protect mice against an otherwise lethal challenge inoculum of 10(3) bloodstream-form trypomastigotes. The injection route used for immunization was shown to have a marked impact on the development of protective immunity. Mice receiving subcutaneous (s.c.) injections of PAR proteins had reduced bloodstream parasitemias and showed 100% survival following challenge. In contrast, mice immunized via the intraperitoneal (i.p.) route developed parasitemia levels equivalent to those of unimmunized controls and did not survive infection. Western blotting (immunoblotting) demonstrated that sera from both i.p. and s.c. immunized mice reacted specifically with PAR proteins; however, the antibody titer of the i.p. immunized mice was approximately 64-fold greater than that of the s.c. immunized mice, suggesting that the protective response in the s.c. immunized mice is cell mediated rather than humoral.

The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

The trypomastigote specific surface antigens of Trypanosoma cruzi are encoded by a supergene family which includes the TSA family. The TSA family is characterized by the presence of a 27-bp tandem repeat array in the coding region. Here, we report the characterization and analysis of the three TSA family members in the Esmeraldo strain of the parasite. In this strain 2 distinct telomeric members are expressed abundantly as 3.7-kb mRNAs, while the remaining member is located at an internal chromosomal site and is expressed at less than 2% of the level seen for the telomeric members. Based on hybridization to DNA separated by PFGE, 3 chromosomes of sizes 1.8 Mb, 0.98 Mb, and 0.90 Mb each contain one of the telomeric members. In addition, the two smaller chromosomes also contain the single internal member. Since both chromosomes contain similar TSA family members, and vary only slightly in size, we suggest that they are homologues. Comparisons of the nucleotide sequences of the different members of the family show that the internal gene differs from the telomeric genes primarily in sequences found 3' of the repeat array. These comparisons also reveal that the three genes are analogous, supporting the hypothesis that short segments between the family members are exchanged by gene conversion events. We propose that similar conversion events between members of different gene families may generate some of the diversity found within the supergene family.

The previously identified major protein components of the paraflagellar rod in Trypanosoma cruzi, PAR 1 and PAR 2, were analyzed to determine if they are distinct proteins or different conformations of a single polypeptide as has been suggested for other trypanosomatids. Amino acid sequence analysis showed PAR 1 and PAR 2 to be two distinct polypeptides. Antibodies specific against either PAR 1 or PAR 2 were shown to each react with a distinct band in Western blots of paraflagellar isolates of T. cruzi and other trypanosomatids if rigorous protease inhibition was used. The PAR 2 message was isolated and characterized by Northern blot and nucleic acid sequence analysis. Preliminary analysis of the PAR 2 gene indicates that PAR 2 is a member of a multigene family with all members residing on a single chromosome.

The X-linked gene LSP1-alpha of Drosophila melanogaster, expressed in the third larval instar, does not exhibit dosage compensation at its normal locus but does compensate when it is relocated to ectopic sites on the X chromosome. A transcription unit designated L12, which is active in the second larval instar and capable of encoding a putative protein of 28.5 kDa, lies immediately downstream from LSP1-alpha. We have determined that L12 is dosage compensated by measuring the steady-state level of its transcript in male and female larvae. The difference in response of these two adjacent genes should be taken into consideration when models of the mechanism of dosage compensation are formulated.

In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes.

A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.

A hybridoma cell line was derived from spleen cells of B6D2 mice infected with the Peru strain of Trypanosoma cruzi. The monoclonal antibody produced by this hybridoma, designated mAb20H1, reacts exclusively with molecular components of trypomastigotes, the infective form of the parasite. The results of indirect immunofluorescence and of immunoelectron microscopy with gold-tagged antibodies indicate that the 20H1 antigen is restricted to the surface of the part of the flagellum in contact with the cell body and to the surface of the cell body in the immediate vicinity of this organelle. Western blot analysis showed that the 20H1 antigen consists of four to five different molecules with sizes between 34 and 41 kDa, and that these molecules are glycoproteins with affinity for concanavalin A. In other strains of T. cruzi, mAb20H1 reacts with glycoproteins with apparent sizes that range between 37 and 43 kDa in the CL, Esmeraldo and Y strains, and between 41 and 45 kDa in the Silvio strain.

In studies of the resistance of inbred mice to infection with Trypanosoma cruzi Peru, mouse strain B10.S was the only strain which survived the infection resulting from the inoculation of 10(3) trypomastigotes. This is the only inbred mouse strain studied to survive infection. To investigate the effect of the H-2 haplotype on survival, C57BL/10 congenic mouse strains bearing H-2S recombinant haplotypes and mouse strains A.SWSn/J and SJL/J were tested for their ability to overcome the T. cruzi infection. None of the recombinant strains tested, including B10.S(7R), B10.S(8R), B10.S(9R), and B10.HTT, survived the infection, indicating that at least two or more regions of the H-2 locus must be H-2S to ensure survival. Strains A.SWSn/J and SJL/J with the H-2S haplotype did not survive, indicating that the genetic background outside the H-2 complex also influences survival. The congenic F1 hybrid (C57BL/10 X B10.S) F1 exhibited intermediate survival levels when compared with the parental strains, indicating that H-2S survival is affected by gene dosage. The F1 hybrid strain [B10.S(7R) X B10.S(8R)]F1, which possesses the complete H-2S haplotype in the trans configuration, did not survive T. cruzi infection, suggesting that H-2S-mediated survival does not operate by trans complementation.

Velocity sedimentation studies of RNA of Sarcophaga bullata show that the major rRNA species have sedimentation values of 26S and 18S. Analysis of the rRNA under denaturing conditions indicates that there is a hidden break centrally located in the 26S rRNA species. Saturation hybridization studies using total genomic DNA and rRNA show that 0.08% of the nuclear DNA is occupied by rRNA coding sequences and that the average repetition frequency of these coding sequences is approximately 144. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA was determined by the examination of the structure of rRNa:DNA hybrids in the electron microscope. Long DNA strands contain several gene sets (18S + 26S) with one repeat unit containing the following sequences in order given: (a) An 18S gene of length 2.12 kb, (b) an internal transcribed spacer of length 2.01 kb, which contains a short sequence that may code for a 5.8S rRNA, (c) A 26S gene of length 4.06 kb which, in 20% of the cases, contains an intron with an average length of 5.62 kb, and (d) an external spacer of average length of 9.23 kb.

Monoclonal antibodies reactive with the surface antigens of the Peru strain of Trypanosoma cruzi were analyzed by Western blots and immunofluorescence assays to determine their reactivity with three life cycle stages and five strain isolates of T. cruzi. One monoclonal antibody, 7.6, recognized a 68-kilodalton (kDa) polypeptide in Western blots of Peru strain trypomastigotes, epimastigotes, and amastigotes. A 68-kDa polypeptide was also detected by monoclonal antibody 7.6 in trypomastigotes of the CL and Y strains and in the clonal isolates Esmeraldo clone 3 and Silvio X10 clone 1. Positive immunofluorescence results were obtained for all life cycle stages of the five strains that were reacted with monoclonal antibody 7.6, thus indicating that the antigen recognized by monoclonal antibody 7.6 is universally present in all T. cruzi strains tested. In contrast, monoclonal antibody 4.2 reacted with a polypeptide doublet of 90 and 105 kDa in Western blots of Peru strain trypomastigotes, but it did not detect these antigens in epimastigotes or amastigotes. The same polypeptide doublet of 90 and 105 kDa was also detected in Western blots of Y strain trypomastigotes; however, no bands were detected in blots of strain CL or isolate Silvio X10 clone 1 trypomastigotes. In blots of Esmeraldo clone 3 trypomastigotes, a single band of 130 kDa was detected by monoclonal antibody 4.2. In immunofluorescence assays of monoclonal antibody 4.2, positive reactions were obtained only with trypomastigotes of Peru, Y, and Esmeraldo clone 3 strains. Thus, monoclonal antibody 4.2 recognizes a trypomastigote-specific antigen which is not universally present on all strains of T. cruzi.

Glucose-6-phosphate dehydrogenase (G6PD) has a major role in NADPH production and is found in almost all cell types. The structural gene for G6PD is X-linked in Drosophila melanogaster, as it is in most eukaryotic organisms, and due to its ubiquitous expression, it can be considered a typical 'housekeeping' gene. Here we present the complete nucleotide (nt) sequence of G6PD cDNAs as well as the genomic copy of the G6PD gene. The G6PD gene has three introns so that the protein-coding region is divided into four segments. The 5'-end of mature G6PD mRNA is located 289 +/- 1 nt upstream from the start codon. The sequence upstream from the transcription start point is G + T-rich and contains no commonly found transcription regulatory elements, such as a TATA box or GGGCGG sequence. D. melanogaster G6PD is 65% homologous with the human G6PD protein but has no homology with the human sequence for the first 42 amino acid residues. The G6PD gene was shown to be active when transduced to autosomal positions. For each transformant, G6PD activity in both male and female adults was not significantly different, indicating that the transduced gene, unlike the resident G6PD, is not dosage-compensated in males.

A recombinant cDNA library constructed in the expression vector lambda gtll using mRNA from the trypomastigote stage of Trypanosoma cruzi was screened with two monoclonal antibodies that have been shown to react with a 105 kDa and a 90 kDa surface antigen in trypomastigotes of the Peru and Y strains of T. cruzi. One recombinant lambda phage, designated Tcc-20, was reactive to both monoclonals. The beta-galactosidase/T. cruzi hybrid protein encoded in Tcc-20 is recognized by the monoclonal antibodies and by serum antibodies from mice infected with strains of T. cruzi which contain the 90 kDa antigen. Antibodies immunoselected from serum of mice infected with the Peru strain by adsorption to Tcc-20 fusion protein react specifically with a 90 kDa polypeptide in trypomastigote but not epimastigote lysates of T. cruzi. The mRNA complementary to the DNA insert in Tcc-20 is present only in those stages and strains of T. cruzi which express the 90 kDa surface antigen. These characteristics are strong evidence that the T. cruzi DNA fragment cloned into Tcc-20 encodes a portion of the 90 kDa surface antigen. The gene(s) which encodes this polypeptide is shown to be present in approximately 20 copies per haploid genome and most, and possibly all, of the copies are found in a tandemly linked multigene family.

Total polysomal RNA and polyadenylated mRNA from third instar larvae, pupae, and adults of D. melanogaster were hybridized in vast excess to labeled single-copy DNA in order to measure the sequence complexity of each RNA population. Then, to measure the sequence homology between the populations, each was hybridized to DNA enriched for messenger coding sequences in third instar larvae and to DNA depleted of these sequences. Our results show that a similar number of genes, approximately 16,000, is expressed in larvae, pupae, and adults, and that only one-third of these is expressed as polyadenylated mRNA. Further, the composition of both polyadenylated and nonpolyadenylated mRNA classes is shown to change very little between these three stages of development. Finally, the head of adult Drosophila is shown to contain 11,000 RNA species, approximately 70% of the number contained in the entire adult. © 1981.

The expression of 20 cloned DNA sequences, selected on the basis of the prevalence of their transcripts in Drosophila adult head, was examined during Drosophila development. The amount of polyadenylated RNA complementary to these clones was measured in the cytoplasm of 0- to 20-hr embryos and on the polysomes of third instar larvae and 5- to 7-hr pupae. Four patterns of developmental expression were observed among the 20 clones, including 6 clones whose transcripts are prevalent only on polysomes of the adult head. Multiple genes closely clustered within the DNA of individual clones were shown to be expressed coordinately during development in some cases and differentially in others. © 1982.

The surface polypeptides of epimastigotes and tissue culture-derived trypomastigotes of Trypanosoma cruzi have been isolated free of most cytosolic components by use of the 2-iminobiotin-avidin interaction. Polypeptides of the trypomastigote stage obtained by this technique are recognized by serum antibodies from Chagasic patients and T. cruzi-infected mice. These polypeptides have been used as the detecting antigen for the identification of hybridoma cells producing monoclonal antibodies against the surface proteins of the trypomastigote stage of T. cruzi. These experiments document a practical approach for obtaining T. cruzi surface proteins in sufficient quantity and purity for use in immunological studies.

Cot and chemical analysis show that the haploid genome size of Thermobia domestica is 3-4x10(9) nucleotide pairs. Of this DNA 33% is single copy sequences and 67% is repetitive sequences. The repetitive sequences are predominantly 300 nucleotides in length and are interspersed among the single copy sequences in a short period interspersion pattern similar to that observed in Xenopus and many other higher eucaryotes. The DNA sequence organization observed in Thermobia is compared with that of other more highly evolved insects.

The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 x 10(8) nucleotide pairs (2.8 x 10(-13) g) and a kinetoplast DNA content of 4.9 x 10(7) nucleotide pairs (5.4 x 10(-14) g). Reassociation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 x 10(3) present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments. By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A)+-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNA's of different abundancy levels: two sequences which occur ca. 3000 per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1-2 times per cell.

Comparative screening of a library of cloned Drosophila DNA with polyadenylated mRNA from Drosophila adult head and adult body identified 20 cloned sequences expressed more abundantly in the tissues of the head than in other tissues. Quantitation using a "dot blot" hybridization assay demonstrated that the DNA sequences are expressed on adult head polysomes from 3 to 177 times more abundantly than on body polysomes, and their transcripts represent from 0.05 to 0.66% of the polyadenylated mRNA mass of the head. The steady-state nuclear RNA concentrations of four species were determined to be from 13 to 48 times greater in head nuclei than in body nuclei, an indication that their expression is controlled at least in part at the transcriptional level. The chromosomal locations of all 20 head-specific clones were identified by in situ mapping, and no distinct clustering was observed. However, four of the clones were shown by Southern and Northern blot analysis to contain multiple RNA coding sequences. The genes in these tightly clustered sets were observed to be expressed simultaneously in some cases and differently in others. © 1982.

To investigate the molecular basis of dosage compensation in Drosophila, a recombinant lambda phage containing the Drosophila melanogaster glucose-6-phosphatase dehydrogenase (G6PD) gene was isolated by differential screening of a Drosophila genomic lambda library with poly(A)+RNA obtained from polyribosomes enriched for or depleted of G6PD mRNA sequences. Of 44 000 plaques screened, a single phage, lambda DmG21, showed hybridization with the enriched poly(A)+RNA but not the depleted one. Confirmation that the Drosophila DNA fragment cloned in lambda DmG21 contains the G6PD gene sequence is based on the following observations. lambda DmG21 DNA shows hybridization only to the 18D region of the salivary gland X-chromosome, which is the known cytological locus for the G6PD gene. In vitro translation of the poly(A)+mRNA selected by hybridization to lambda DmG21 DNA sequences shows a polypeptide product of apparent Mr 55 000, identical to that of the monomeric unit of G6PD. When the putative coding sequence of G6PD is cloned into the expression vector lambda gt11, recombinant plaques are recognized by anti-G6PD immunoglobulin. A transcriptional map of the G6PD gene shows that it is divided into two exons, 0.9 kb (exon I) and 1.8 kb (exon II) long, which are separated by a 2.4-kb intron. The G6PD mRNA is 2.0 kb in length and the steady-state level of the mRNA is similar in both sexes. Measurement of the copy number of the G6PD gene in males and females shows the gene to be present once per X-chromosome in both sexes. No amplification of the gene sequence was observed in males. These results are, therefore, in agreement with the previous suggestion that dosage compensation is the result of enhanced transcription of X-linked genes in males.

The sequence complexity of nuclear total RNA and nuclear poly(A)+RNA from Drosophila third-instar larvae was determined by hybridization of these RNAs to labeled single-copy DNA. At saturation, the nuclear poly(A)+ - and total RNA hybridized to 11% and 22.5% of the single-copy DNA, respectively. The increase in complexity of nuclear total RNA over that observed for nuclear poly(A)+RNA indicates the presence of a discrete class of nonoadenylylated nuclear RNA molecules. The relationship between DNA sequences coding for nuclear RNA and mRNA was then determined by hybridization of nuclear total and poly(A)+RNA to DNA enriched for mRNA coding sequences. The results of these studies show that those single-copy DNA sequences that are represented in either the poly(A)+ - or poly(A)- mRNA population are transcribed into RNA molecules that appear in the nuclear poly(A)+RNA population.

A 320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analysis of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the terminal repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.

Null activity alleles of the structural genes for glucose-6-phosphate dehydrogenase and xanthine dehydrogenase in Drosophila melanogaster have no or little measurable effect on the viability of mutant flies. In contrast, double mutant combinations lacking both enzyme activities result in lethality. This observation highlights a physiological interaction between metabolic pathways heretofore considered to be independent. © 1988.

The surface proteins of six stages of Trypanosoma cruzi were labeled by Iodogen-catalized surface iodination and analyzed by one and two dimensional polyacrylamide gel electrophoresis. These stages included bloodstream trypomastigotes, culture-form trypomastigotes, amastigotes, staphylomastigotes, epimastigotes, and metacyclic trypomastigotes. Antigens recognized by serum antibodies were detected by Western blotting against serum from mice hyperimmunized against bloodstream trypomastigotes. Bloodstream trypomastigotes, culture-form trypomastigotes and staphylomastigotes contained several surface proteins of molecular weight (Mr) 90 000 and isoelectric points (pI) between 5.0 and 7.5. Western blotting reveals that at least two proteins of 90 000 Mr represent the major antigens seen on bloodstream trypomastigotes, culture-form trypomastigotes, staphylomastigotes and amastigotes. However, a 90 000 Mr protein was not detected by either Western blotting or surface iodination on epimastigotes or metacyclic trypomastigotes. The major surface proteins on these latter two stages were represented by several 72 000 Mr proteins with pI values between 5.2 and 5.8. An interesting result of this survey is that a 90 000 Mr surface antigen was present on staphylomastigotes, a stage which can be grown in cell free medium.

OBJECTIVE: Pulse contour cardiac output (PCCO) analysis is a technique for continuous cardiac output (CO) monitoring through an arterial catheter after calibration by transpulmonary thermodilution (TPTD). Studies in adults show good correlation with pulmonary artery thermodilution (PATD) CO. Data are limited in children and patients with hemodynamic instability. The objective was to determine whether TPTD CO and PCCO analysis correlate with PATD CO in a piglet model of severe hemorrhagic shock. Mixed venous oxygen saturation (SVO2) was also compared with PATD CO. DESIGN: Prospective animal study. SETTING: University animal research laboratory. SUBJECTS: Domesticated piglets, 24-37 kg. INTERVENTIONS: Hemorrhagic shock was created by graded hemorrhage in anesthetized piglets. Hemorrhage was initiated to achieve mean arterial pressure plateaus of 60, 50, 40, 30, and 20 mm Hg. MEASUREMENTS AND MAIN RESULTS: CO was measured by PATD and simultaneously with two femoral artery PCCO catheters. At each mean arterial pressure plateau, one PCCO catheter was recalibrated by TPTD; the other catheter was not recalibrated during hemorrhage. TPTD CO, PCCO measurements from each catheter, and SVO2 were compared with PATD CO at each mean arterial pressure level. TPTD CO and recalibrated PCCO showed excellent correlation (r2 = .96 and .97) and small bias (+0.11 and +0.14 L/min), respectively, compared with PATD. Without recalibration, PCCO measurements were not accurate during rapid hemorrhage (r2 = .22). SVO2 decline did not correlate as well with PATD CO (r2 = .69). CONCLUSIONS: TPTD CO and recalibrated PCCO analysis correlate well with PATD CO in this severe hemorrhagic shock model. The mean difference is small (<0.15 L/min) and is not clinically significant. With rapid changes in blood pressure or intravascular volume, PCCO is not accurate unless recalibrated by TPTD CO. SVO2 did not correlate well with CO in this model. [ABSTRACT FROM AUTHOR]

Background: Seroprevalence studies are the standard for disease surveillance, and serology determined eligibility for the first dengue vaccine. Expanding flavivirus co-circulation and vaccination complicate testing. We evaluate the accuracy of a common dengue virus serological assay, examine immunity to non-dengue flaviviruses as a contributor to decreased performance, and assess whether alternative cut points may improve assay performance., Methods: Children (n = 770) aged 2-9 years in Kampong Speu, Cambodia were enrolled in a prospective longitudinal study, and PanBio indirect dengue virus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was performed. Plaque reduction neutralization tests (PRNTs) using dengue viruses were performed on a subset to assess the accuracy of the IgG ELISA, and PRNTs with Zika, Japanese encephalitis, and West Nile viruses evaluated immunity to non-dengue flaviviruses. Receiver operating curve analysis identified an alternative cut point to improve IgG ELISA accuracy., Results: The dengue IgG ELISA had a lower specificity than previously reported (58% vs 93%-100%). Of those with false-positive IgG results, 46% had detectable neutralizing antibodies against other flaviviruses including 14% against West Nile virus. A higher IgG cut point improved the test accuracy in this population., Conclusions: Physicians and public health authorities should be alert for West Nile in Cambodia. Immunity to non-dengue flaviviruses can impact dengue surveillance., Clinical Trials Registration: NCT03534245., Competing Interests: Potential conflicts of interest. J. E. M. is now a Clinical Franchise Leader of Influenza in Vaccines Research and Development at Sanofi Pasteur. The work reported in this manuscript was conducted prior to her switching positions. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Type-H capillary endothelial cells control bone formation during embryogenesis and postnatal growth but few signalling mechanisms underpinning this influence have been characterised. Here, we identify a highly expressed type-H endothelial cell protein, Clec14a, and explore its role in coordinating osteoblast activity. Expression of Clec14a and its ligand, Mmrn2 are high in murine type-H endothelial cells but absent from osteoblasts. Clec14a -/- mice have premature condensation of the type-H vasculature and expanded distribution of osteoblasts and bone matrix, increased long-bone length and bone density indicative of accelerated skeletal development, and enhanced osteoblast maturation. Antibody-mediated blockade of the Clec14a-Mmrn2 interaction recapitulates the Clec14a -/- phenotype. Endothelial cell expression of Clec14a regulates osteoblast maturation and mineralisation activity during postnatal bone development in mice. This finding underscores the importance of type-H capillary control of osteoblast activity in bone formation and identifies a novel mechanism that mediates this vital cellular crosstalk., (© 2024. The Author(s).)