A Primary Linkage Map of the Porcine Genome Reveals a Low Rate of Genetic Recombination

A comprehensive genetic linkage map of the porcine genome has been developed by typing 128 genetic markers in a cross between the European Wild Boar and a domestic breed (Large White). The marker set includes 68 polymerase chain reaction-formatted microsatellites, 60 anchored reference markers informative for comparative mapping and 47 markers which have been physically assigned by in situ hybridization. Novel multipoint assignments are provided for 54 of the markers. The map covers about 1800 cM, and the average spacing between markers is 11 cM. We used the map data to estimate the genome size in pigs, thereby addressing the total recombination distance in a third mammalian species. A sex-average genome length of 1873 +/- 139 cM was obtained by comparing the recombinational and physical distances in defined regions of the genome. This is strikingly different from the length of the human genome (3800-4000 cM) and is more similar to the mouse estimate (1600 cM). The recombination rate in females was significantly higher than in males.     

Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.     

Outbreak of Giardiasis: Effect of a New Antiflagellate Drug,Tinidazole

We describe here an intensive outbreak of mostly symptomatic (90%) Giardia lamblia infestation in a Swedish student group visiting the U.S.S.R. A new antiflagellate drug, ethyl (2-(2-methyl-5-nitro-1-imidazolyl)ethyl) sulphone, tinidazole, Pfizer, was given in a dosage of 150 mg twice daily for seven days to 10 healthy volunteers and to 24 students infested with G. lamblia. The drug was found to be effective in curing giardiasis and in eradicating G. lamblia from the intestinal tract. All the students with symptomatic giardia infestation became free from gastrointestinal disturbance, usually soon after treatment was started. None of the 24 students had G. lamblia in their stools after tinidazole treatment was discontinued or at follow-up. No side effects of the drug were seen and all of the subjects tolerated it very well.     

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations or membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle.If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation.It is suggested that fragmentation of paired membranes followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.     

Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Functional maturation of human T lymphocytes is accompanied by changes in the G-protein pattern

The putative guanine nucleotide binding (G)-protein involved in transduction of signals from the TCR/CD3 complex has not been identified. We have used a UV-photoaffinity labeling technique to covalently attach [alpha-32P]GTP to human lymphocyte and thymocyte membrane proteins. Ten bands specifically labeled with [32P]GTP were detected by SDS-PAGE and autoradiography in T lymphocyte membranes. Among these, a 40-kDa protein was identified by immunoblotting as the alpha-subunit of the adenylate cyclase-inhibiting G-protein, Gi, and two proteins of 44 and 46 kDa were identified as the alpha-subunits of adenylate cyclase stimulating G-protein (Gs). These proteins also served as substrates for ADP-ribosylation by pertussis toxin and cholera toxin, respectively. Comparison of GTP-labeled membrane proteins from immature and more mature thymocytes and blood T lymphocytes, revealed that bands of 26, 30, 34, 40, 44 and 46 kDa were absent or weakly labeled in immature thymocytes, intermediate in mature thymocytes, and strongest in blood T cells. Similar increases were seen in ADP ribosylation of the substrates for pertussis, cholera, and botulinum C3 toxin. However, corresponding quantitative changes in Gi and Gs were not detected by immunoblotting, which suggests that the increased labeling is caused by enhanced affinity of the proteins for GTP rather than by increased amount of protein during thymic maturation. A concomitant maturation of GTP-induced cAMP production was seen in the cell populations, but no such change occurred in direct activation of adenylate cyclase by forskolin. The changes in some (but not all) GTP-binding proteins during acquisition of immunocompetence indicates their importance in T lymphocyte physiology.     

Male nest building but not display behaviour directly influences mating success in the polygynous Red Bishop,Euplectes orix

Previous studies of the highly polygynous and strikingly sexually dimorphic Red Bishop, Euplectes orix (Ploceinae, weaverbirds), have suggested random female settlement patterns and no correlates of male reproductive success except the number of nest frames (‘cock's nests’) built by the male. Although this confirms the central role of the nest in weaverbird courtship it also contrasts with demonstrated sexual selection on male morphology and behaviour in several closely related Euplectes species. Two major aspects of male sexual advertising have not been included in previous studies; display behaviour and territory size. In this study we use multivariate selection analysis, with the number of active nests in a territory as the fitness measure, to identify direct and indirect sexual selection on male sexual behaviour, territory size and nest building. In accor–dance with previous findings, mating success was not strongly skewed among the territorial males, and females appeared to settle randomly with respect to available nests. The number of cock𠀧s nest built was the only determinant of male breeding success, even when controlling for male display activity and territory size. We argue that, despite their conspicuous sexual dimorphism, females settle independently of both male or territory quality, and that variance in male reproductive success is a consequence of male–male competition.     

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.     

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.