Identification and genetic mapping of Xenopus TAP2 genes.

The amphibian Xenopus laevis is one non-mammalian vertebrate in which the major histocompatibility complex (MHC) has been analyzed extensively. Class IIbeta, class Ia, LMP2, LMP7, HSP70, C4, Factor B, and Ring3 genes have been identified and mapped to the MHC. Here, we report the isolation of a transporter associated with antigen processing (TAP) gene, TAP2, and demonstrate its linkage to the MHC. While the ATP-binding region of Xenopus TAP2 is highly conserved in evolution, amino acid identity to other vertebrate TAP proteins was not detected in the N-terminal region. Segregation analysis of 34 individuals from two families showed exact restriction fragment length polymorphism matching between the MHC class Ia gene and the one TAP2 gene demonstrating linkage conservation since the mammalian/amphibian divergence approximately 350 million years ago. In addition, one non-MHC-linked TAP2-hybridizing fragment was detected in approximately half of the individuals tested. Interestingly, TAP2 allelic lineages appear to match those of LMP7 and classical class I, which previously were categorized into two highly divergent groups that emerged at least 60 million years ago. Similar to LMP7 and class Ia,TAP2 is expressed ubiquitously with highest levels in intestine and spleen.

Co-evolution of rat TAP transporters and MHC class I RT1-A molecules.

The genes for rat major histocompatibility complex (MHC) class I molecules are associated either with those for the A allele of the transporter associated with antigen processing (TAP-A), which can transport peptides with basic carboxy-terminal residues, or with those for TAP-B, which cannot [1-5]. To explore whether these associations have a functional basis, we compared the sequences of 13 rat MHC class la RT1-A cDNAs from nine MHC haplotypes. Of seven TAP-A- linked RT1-A molecules, six possess strongly acidic F pockets, and these bind a high proportion of peptides with basic carboxy-terminal residues. The F pockets of TAP-B-linked molecules, by contrast, were more basic. Furthermore, we identified six positions at the 'righthand end' of the peptide-binding groove, at which a majority of TAP-B-linked molecules diverge from the consensus sequence for class la molecules whereas, at these positions, all the TAP-A-linked molecules reflect the consensus sequence. Our results suggest that the linked rat class la and TAP genes have co-evolved to maximize the supply of appropriate peptides to the presenting molecules.

Functional analysis by site-directed mutagenesis of the complex polymorphism in rat transporter associated with antigen processing.

The transporter associated with Ag processing, TAP, is an endoplasmic reticulum resident heterodimeric member of the ATP-binding cassette transporter family. TAP transports short peptides from cytosol to the endoplasmic reticulum lumen for loading into recently synthesized class I MHC molecules. In the rat, two alleles of the TAP2 chain differ in their permissiveness to the transport of peptides with small hydrophobic, polar, or charged amino acids at the C terminus, and this correlates with differences between the peptide sets loaded into certain class I molecules in vivo. We have used segmental exchanges and site-directed mutagenesis to identify the residues in rat TAP2 responsible for differential transport between the two alleles of peptides terminating above all in the positively charged residue, arginine. Of the 25 residues by which the two functional TAP2 alleles differ, we have localized differential transport of peptides with a C-terminal arginine to two adjacent clusters of exchanges in the membrane domain involving a total of five amino acids. Each cluster, transferred by site-directed mutagenesis from the permissive to the restrictive sequence, can independently confer on TAP a partial ability to transport peptides with arginine at the C terminus. The results suggest that the permissive TAP2-A allele evolved in at least two steps, each partially permissive for peptides with charged C termini.

Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

The rat MHC haplotype RT1c expresses two classical class I molecules.

Cloning and characterization of classical MHC class I coding sequences of the laboratory rat Rattus norvegicus has been reported so far for only four haplotypes, RT1a, RT1(1), RT1n, and RT1u. In all four cases, only one RT1.A classical class I molecule was found. Here we report that, in contrast, the RT1c haplotype expresses two different classical class I molecules. Using recombinant rat strains, we find that allotypic serologic determinants carried by the two molecules map to the RT1.A region, and so we have named them RT1.A1c and RT1.A2c. Multiple clones of functional cDNAs for each of these two molecules were isolated using a recently developed PCR-based expression-cloning method. Using a panel of 20 RT1.Ac-reactive mAb, we find that six recognize RT1.A1c, seven recognize RT1.A2c, and seven recognize both. We also show that both molecules are recognized and distinguished by primary alloreactive cytotoxic T lymphocytes, and that they correspond to identifiable and distinct molecular species in cells that express RT1c naturally. These data all concur to demonstrate that the RT1.Ac region carries two different loci, each of which encodes a functional classical class I molecule.

Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar.

The bovine IgG1 and IgG2 isotypes exhibit large differences in effector functions. To examine the structural basis for this, the 12-domain structures of IgG1 and IgG2 were investigated by pulsed neutron scattering using a recently developed camera LOQ. This method reports on the average relative disposition in solution of the Fab and Fc fragments in IgG. The radii of gyration (RG) were found to be similar at 5.64 and 5.71 nm for IgG1 and IgG2 respectively in 100% 2H2O buffers. The two cross-sectional radii of gyration (RXS) were also similar at 2.38-2.41 and 0.98-1.02 nm. Similar values were obtained for porcine IgG. Both bovine IgG1 and IgG2 possess similar overall solution structures, despite sequence differences at the hinge region at the centre of their structures. An automated computer survey of possible IgG structures was developed, in which coordinates for the two Fab fragments were displaced in a two-dimensional plane relative to those of the Fc fragment in 0.25 nm steps. The scattering curves calculated from these structures were found to be sensitive to relative displacements of the three fragments, but not on their rotational orientation about their longest axes. Good agreement with the solution scattering data was obtained with a planar IgG model in which the C-terminus of the CH1 domain of Fab was 3.6 nm from the N-terminus of Fc in both IgG1 and IgG2, with a precision of 0.7 nm. Energy refinement showed that this spatial separation is compatible with the hinge sequences of bovine IgG1 and IgG2. The results show that multidomain protein structures can be modelled using LOQ data, and that a long hinge sequence does not necessarily reflect a large distance between Fab and Fc. The steric accessibility of Fc sites for interactions with cell-surface Fc receptors and C1q of complement is shown to be generally similar for IgG1 and IgG2, and the difference in effector function between IgG1 and IgG2 is probably based on deletions in the IgG2 hinge sequence.

Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule.

Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.

MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters.

The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system. Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER). In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism. Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC. Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance. Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway. Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.

Vaccination of young lambs by means of a protein fraction extracted from adult Haemonchus contortus.

Lambs aged 48-150 days which had been injected with extracts enriched in a functional antigen (contortin) obtained from adult Haemonchus contortus developed specific circulating antibodies and were less susceptible to haemonchosis when challenged 1 month later with a single dose of 20,000-25,000 infective 3rd-stage larvae. Sera from 18 out of a total of 19 lambs injected with the extract contained precipitating antibodies to 2-5 components of the extract. None of these lambs died. The one extract-injected lamb which did not develop antibodies and 9 of the 13 lambs used as controls died of haemonchosis. The average weight of worms recovered was 1.45 g from the immune lambs and 5.72 g from the non-immune lambs.

The use of faecal egg counts for estimating worm burdens in sheep infected with Haemonchus contortus.

Clun forest sheep, worm-free from birth, were given a single dose of 20 000 infective larvae of Haemonchus contortus. The total number of eggs/day in the faeces was recorded for 21 infections and data on population size, sex ratio and individual worm development were collected from 76 sheep. The female to male ratio was 1.28 +/- 0.07 (S.E.). The relation between increase in worm size and uterine egg content was linear. The number of eggs present in the uteri was found to be an accurate measure of eggs passed. It was shown that the daily faecal egg output is related to total parasite weight and is not a measure of the number of individuals present.

The development, composition and maintenance of experimental populations of Haemonchus contortus in sheep.

Clun Forest sheep, aged between 3 and 18 months and worm-free from birth, were given a single dose of 25 000 infective larvae of the nematode Haemonchus contortus. The host animals were killed between 4 and 100 days after infection and the nematode populations were examined to determine size and composition. The relation between worm body length, dry weight and age was studied and growth curves were constructed. Variations in the sex ratio for infections of different ages were noted. No evidence was found for a relation between rate of growth and population density. The rate of expulsion was determined and its variability discussed.

A new technique for the extraction of Nematodirus battus eggs from sheep faeces.

Details are given of a method for the separation of the eggs of the nematode Nematodirus battus from sheep faeces. Faecal pellets obtained from worm-free lambs experimentally infected with N. battus were homogenized and passed through a graded series of metal sieves. The material retained in a 53 mum aperture sieve was transferred to a cylindrical column and the eggs were cleaned and separated from other suspended material by controlled differential flotation using tap water. A model experiment indicated that, within limits, the principle held for any size of tower. The density distribution for eggs at the morula stage of development was determined. Suggested flow rates for a specified tower are given.

The glucose metabolism of adult Ostertagia circumcincta, in vitro.

Adult Ostertagia circumcincta from freshly killed sheep were incubated at 39 degrees C in a medium containing inorganic salts, antibiotics and D-[U-14C] glucose. The worms appeared healthy even after incubation for as long as 72 h. All the radioactivity was recovered either within the worms or in the incubation vessel in the form of metabolic products or unmetabolized glucose. Incubations were carried out at low oxygen tension except for those in which CO2 was measured. These were either aerobic or anaerobic. In terms of both quantity and radioactivity the main metabolic products of glucose were CO2, propan-1-ol, acetate and propionate. Smaller amounts of ethanol, lactate and succinate were formed. The results are compared with those found for the similar nematode Haemonchus contortus.

Observations on the development of Haemonchus contortus in young sheep given a single infection.

Worm free lambs 2-4 months old were given a single infection of 50,000 Haemonchus contortus larvae. The changes in packed red cell volume were compared with those of uninfected sheep and the infected sheep were divided into two groups according to the rate of change of packed cell volume. In group 1, packed cell volume fell sharply from the 10th day of infection and the sheep eventually died. In group 2, the fall was less marked and the packed cell volume returned to normal after about 40 days. The rates of increase of parasite body length and dry weight were compared for the two groups. Parasites in group 1 sheep grew more slowly than their group 2 contemporaries. Accurate planning of metabolic studies on H. contortus was made possible by using this information to predict the course of an infection and the size of the worms. Parasite development was also measured in sheep for which the packed cell volume was not recorded. Computer analysis showed that adult worm length increase did not follow a simple growth pattern starting from the last ecdysis, but consisted of a rapid elongation followed by a simple negative exponential increase. Dry weight increase also followed a negative exponential during the second phase. Measurements of dry weight as a percentage of wet weight indicated that the rapid elongation was possibly linked with cell enlargement by the uptake of water.