Incomplete offspring sex bias in Australian populations of the butterfly Eurema hecabe.

Theory predicts unified sex ratios for most organisms, yet biases may be engendered by selfish genetic elements such as endosymbionts that kill or feminize individuals with male genotypes. Although rare, feminization is established for Wolbachia-infected Eurema butterflies. This paradigm is presently confined to islands in the southern Japanese archipelago, where feminized phenotypes produce viable all-daughter broods. Here, we characterize sex bias for E. hecabe in continental Australia. Starting with 186 wild-caught females, we reared >6000 F1-F3 progeny in pedigree designs that incorporated selective antibiotic treatments. F1 generations expressed a consistent bias across 2 years and populations that was driven by an ~5% incidence of broods comprising ⩾80% daughters. Females from biased lineages continued to overproduce daughters over two generations of outcrossing to wild males. Treatment with antibiotics of differential strength influenced sex ratio only in biased lineages by inducing an equivalent incomplete degree of son overproduction. Brood sex ratios were nevertheless highly variable within lineages and across generations. Intriguingly, the cytogenetic signature of female karyotype was uniformly absent, even among phenotypic females in unbiased lineages. Molecular evidence supported the existence of a single Wolbachia strain at high prevalence, yet this was not clearly linked to brood sex bias. In sum, we establish an inherited, experimentally reversible tendency for incomplete offspring bias. Key features of our findings clearly depart from the Japanese feminization paradigm and highlight the potential for more subtle degrees of sex distortion in arthropods.

A search for messenger RNA molecules bearing immunoglobulin VH nucleotide sequences in T cells.

Expression of VH-coded mRNA molecules in T cells, antigen-specific T cell lines, or T cell hybridomas was not detected using four different VH DNA probes under conditions that permitted cross-hybridization between distantly related VH genes. In contrast, VH gene expression was readily detected in two B cell lymphomas and in splenic B cells. Less than one molecule per cell of RNA, exactly complementary to the DNA probes used, would have been detected in these T cell populations. The results thus seriously question the proposition that T cells use the B cell VH repertoire to code for antigen receptors.

Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes.

Immunoelectron microscopy with protein A gold has been used to determine the subcellular location of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. RESA was associated with dense vesicles presumed to be micronemes within merozoites. RESA was not detected on the surface of merozoites but was located at the membrane of erythrocytes infected with ring-stage parasites. RESA within merozoites was largely soluble in the nonionic detergent Triton X-100, but was insoluble in this detergent when associated with the erythrocyte membrane.

Keratin synthesis during development of the embryonic chick feather.

The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.

Chromosomes of malaria parasites.

The advent of pulsed field gradient electrophoresis has proved remarkably useful for studying chromosomes of the genetically intractable malaria parasite Plasmodium falciparum. Advances include determination of the karyotype, a linkage map and restriction maps of individual chromosomes that enable the ordering of genes. The structural basis underlying a frequently occurring form of chromosome size polymorphism is now understood and other polymorphisms are providing tantalizing clues to the mechanisms underlying drug resistance.

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Third form of the precursor to the major merozoite surface antigens of Plasmodium falciparum.

The precursor to the major merozoite surface antigens of Plasmodium falciparum appears to be encoded by two distinctly different (dimorphic) alleles able to undergo limited recombination. We analyzed 18 previously uncharacterized P. falciparum isolates to test the dimorphic model. All but one, a Thailand isolate, conformed to the dimorphic model, and this isolate conformed to the dimorphic model in all but variable block 2. Sequence analysis revealed that block 2 of isolate CSL2 was a third form. Hence, the dimorphic model is not strictly correct. Recombination between alleles was found only within two conserved blocks near the 5' end of the gene.

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.

Expression of the RESA gene in Plasmodium falciparum isolate FCR3 is prevented by a subtelomeric deletion.

We show here that the Plasmodium falciparum isolate FCR3 does not express the ring-infected erythrocyte surface antigen (RESA). This is because the 5' end of the RESA gene has been inverted and partly deleted and a telomere has been added to it. We propose a model to explain these events.

Integral membrane protein located in the apical complex of Plasmodium falciparum.

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.

Analysis of Sarcoptes scabiei finds no evidence of infection with Wolbachia.

The endosymbiont Wolbachia has been detected in a range of filarial nematodes and parasitic mites and is known to affect host reproductive compatibility and potentially evolutionary processes. PCR of Wolbachia surface protein (wsp), ftsZ and 16SrRNA genes from individual Sarcoptes scabiei mites obtained from a series of individual hosts, and database searches of an S. scabiei var. hominis EST library failed to detect Wolbachia genes. Therefore, Wolbachia appears not to be involved in the genetic subdivision observed between varieties of host-associated S. scabiei or, involved in the inflammatory disease pathogenesis of scabies unlike its activity in filarial infection.

Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients.

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.

Structure and function of candidate vaccine antigens in Plasmodium falciparum.

Analysis of Plasmodium falciparum antigens expressed in Escherichia coli has identified several different proteins as potential vaccine components. Fragments of one of these antigens, the ring-infected erythrocyte surface antigen (RESA) have been used to protect Aotus monkeys against overwhelming infection with P. falciparum. In this vaccine, trial protection correlated with antibody responses to two of three repetitive sequences in RESA. RESA is released from merozoites and becomes associated with the erythrocyte membrane at the time of merozoite invasion. The 3' repeat structure of RESA encodes a polyacidic sequence that has homology with the N-terminal sequence (cytoplasmic domain) of band 3, the erythrocyte anion transporter. These homologous sequences both include a recognition sequence for a protein tyrosine kinase. It is postulated that RESA disrupts the normal intermolecular interactions of the cytoplasmic domain of band 3 and that phosphorylation of RESA by an erythrocyte membrane kinase in some way regulates this function of RESA.

Affinity purification of human antibodies directed against cloned antigens of Plasmodium falciparum.

A technique has been developed for the affinity purification of antibodies recognizing cloned antigens of the malaria parasite Plasmodium falciparum expressed in bacteria. Adsorbents prepared by coupling bacterial lysates to Sepharose were used to isolate monospecific antibodies from human immune sera. Production of an abundant stable fused polypeptide by the bacteria was not a prerequisite for the success of this approach. Also the procedure permits the characterization of antigens which elicit the production of very low levels of antibodies. Affinity-purified human antibodies were used to characterized the corresponding P. falciparum antigens by immunoblotting and a number of antigens identified in this way illustrate some commonly observed features of P. falciparum antigens. Several of these antibody preparations recognized multiple bands in the electrophoretic patterns. Studies on a number of isolates of P. falciparum indicate that many antigens exhibit size polymorphisms. Production of some antigens was shown to be restricted to particular stages of the asexual blood cycle of the parasite while others appear to be specifically processed during the life cycle. Affinity-purified antibodies have also been used to locate antigens within the infected erythrocyte and to delineate subsets of antibodies recognizing different epitopes of a single antigen.

Sequence diversity of the erythrocyte membrane antigen 1 in various strains of Plasmodium chabaudi.

We have determined the complete genomic sequence of the Plasmodium chabaudi erythrocyte membrane antigen (PcEMA1) in 4 different parasite strains. The gene structure consisted of a short region encoding a signal sequence separated from the main coding region by an intervening sequence. The overall identity of the three P. chabaudi adami deduced protein sequences to their consensus was 100%, 99.8% and 88% for 556KA, DK and DS respectively, with a general pattern of increasing divergence from the N- to the C-terminus. The P. chabaudi chabaudi strain CB was 72% homologous to the P. chabaudi adami consensus sequence. A gene related to PcEMA1, designated PcEMA1-R, has been identified in the genome of P. chabaudi adami but not in P. chabaudi chabaudi. The partial sequence for this gene in P. chabaudi adami strain DS predicts that it could encode a truncated form of PcEMA1, but its status as a pseudogene or an independent, expressed gene has not been resolved.

Large fragments of Plasmodium falciparum DNA can be stable when cloned in yeast artificial chromosomes.

A major problem in cloning large segments of Plasmodium falciparum DNA is the instability of this very A + T-rich DNA in Escherichia coli. We have therefore investigated whether P. falciparum DNA segments over 100 kb in size cloned in the yeast Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) are stable. An analysis of EcoRI fragments adding up to 230 kb, or nearly 1% of the P. falciparum genome, showed that sequences cloned as YACs were indistinguishable from the DNA in the P. falciparum genome, showing neither deletions nor rearrangements. Over 400 YACs have been obtained so far with an average insert size between 110 and 130 kb. Screening the YACs with single-copy probes suggested that most sequences should be represented in a library of less than 10(3) YACs. Hence YACs provide an approach to cloning large segments of P. falciparum DNA.

cDNA clone encoding a high molecular weight antigen of Babesia bovis.

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.

DNA polymorphisms and subpopulations in Babesia bovis.

Independent isolates of Babesia bovis differ by only a limited number of polypeptides, some of which may be important as host protective antigens. Avirulent derivatives of these parasites also differ from their virulent counterparts in only a few polypeptides. To identify genes encoding such polypeptides we have isolated cDNA clones corresponding to poly(A)+ RNAs that are expressed only in certain isolates. For this purpose a cDNA clone library was constructed from poly(A)+ RNA of the K-avirulent isolate (KA). These clones were screened by colony hybridization using [32P]cDNA complementary to poly(A)+ RNA from KA and from virulent isolates, in order to identify clones that selectively hybridize to one cDNA probe. Hybridization of DNA from three clones, designated pK4, pK5 and pK6 to poly(A)+ RNA from various isolates revealed different and complex patterns. The gene represented by clone pK5 appeared to be transcribed predominantly in avirulent parasites. Analysis of genomic DNA by the Southern procedure enabled each isolate to be distinguished and suggested that most isolates are comprised of a heterogeneous mixture of subpopulations. Analysis of genomic DNA from parasites obtained after passage of KA through the tick vector (Boophilus microplus) suggested that a subpopulation was being selected that more closely resembled KV than KA.

A DNA fingerprinting system for the ectoparasite Sarcoptes scabiei.

We describe multiple hypervariable microsatellites that will provide a highly informative genetic marker system for the sarcoptid mite Sarcoptes scabiei. Eighteen positive clones containing the highly repetitive sequence (GA)n were isolated from a partial genomic library of S. scabiei. Ten of these clones were characterised by sequencing and primers were designed from the unique sequences flanking eight microsatellite loci. Genomic DNA was subsequently extracted from individual mites and the repeat blocks were amplified by way of [gamma 33P] ATP end-labelled polymerase chain reaction. Fragment length polymorphisms were revealed in three of the loci when resolved on polyacrylamide sequencing gels. The high levels of allelic variability demonstrated between individual mites enable these three loci to form a DNA fingerprinting system that will be suitable for epidemiological and taxonomic studies both within and between host species.