Founder effect and prevalence of myotonic dystrophy in South Africans: molecular studies.

A high prevalence of myotonic dystrophy (DM) has been described in South African Caucasoid Afrikaans-speaking families in the northern Transvaal. Evidence is presented for a strong founder effect, with a single haplotype occurring on 68% of all Caucasoid DM chromosomes; among the Afrikaans speakers, the proportion was 83%. In addition to this major haplotype, five minor DM haplotypes in the Caucasoids and two minor haplotypes in DM individuals of mixed ancestry were found. All DM chromosomes, however, had a common haplotype core, namely, Alu (ins), HinfI-2 (intron 9), and TaqI-2 (D19S463). We have detected significant linkage disequilibrium between the DM mutation and particular alleles of the extragenic markers D19S112 and D19S207. Significant differences were found in allele and haplotype distributions in the Caucasoid DM and non-DM chromosomes and Negroid non-DM chromosomes. These findings together with the strong association of allele 3 at the D19S63 locus on 93% (14/15) of the South African DM chromosomes suggest that the majority of present-day DM mutations in South African Caucasoids may have originated from a common initial founder who introduced one of the European ancestral mutations.     

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.     

Expression of human IFN-gamma genomic DNA in transgenic mice

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R. Regulation of the transgene paralleled that of the endogenous murine IFN-gamma gene in that: 1) it is not expressed constitutively in any tissue examined thus far, 2) it can be induced in thymus and spleen cells by T cell mitogens, 3) it is not expressed in B cells stimulated by LPS, and 4) it produces normal mRNA and biologically active IFN protein. Whereas expression of the transgene is likely restricted to T cells, we had observed that both fibroblasts and B cell lines could express the same DNA when transfected in vitro; this indicates that in vivo, developmental factors restrict expression of the IFN-gamma gene to T cells. These findings also indicate that the 8.6-kb fragment contains the regulatory elements necessary for normal tissue specific expression in vivo. Moreover, they indicate that the regulatory elements for this gene are completely preserved over the phylogenetic distance separating mouse and man, even though substantial drift has occurred in the structural gene, and probably in the IFN-gamma R as well.     

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.     

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.     

STK25 is a candidate gene for pseudopseudohypoparathyroidism.

We determined the chromosomal location of the mouse gene Stk25, encoding a member of the Ste20/PAK family of serine/threonine kinases, by interspecific backcross analysis. We mapped Stk25 to the central region of mouse chromosome 1 linked to Chrng (formerly Acrg) and En1. This central region of mouse chromosome 1 shares a region of homology with the long arm of human chromosome 2, suggesting that the human homologue of Stk25 would also map to 2q. We proved this prediction of syntenic homology correct by mapping human STK25 to 2q37. Deletion of the 2q37 region has been implicated in the expression of pseudopseudohypoparathyroidism (PPHP), a disease which shares features of the Albright hereditary osteodystrophy (AHO) phenotype. To investigate a pathogenetic relationship between STK25 and PPHP, we carried out fluorescence in situ hybridization (FISH) using an STK25 gene probe and chromosomes from PPHP patients characterized as having small deletions near the distal end of 2q. PPHP patient DNA showed no hybridization to STK25 genomic DNA, indicating that STK25 is contained within the deleted chromosomal region. This finding, in conjunction with previous studies demonstrating the role of Ste20/PAK kinases in heterotrimeric G protein signaling, suggests that STK25 is a positional candidate gene for PPHP.     

A new approach to understanding kinetic cooperativity when the hill coefficients are less than 2

Dihydrofolate reductase from chicken liver has a single sulfhydryl group which reacts stoichiometrically and specifically with a wide variety of organic mercury compounds to yield an enzyme derivative which exhibits up to 10-fold the activity of the unmodified form when measured at pH 6.5, the optimum for the modified enzyme. The sulfhydryl group is apparently not at the active site since a 25-fold excess of either major cosubstrate, dihydrofolate or TPNH, affects neither the rate nor extent of the modification reaction. The reaction is essentially instantaneous and yields an enzyme with altered kinetic properties for all the substrate pairs examined (TPNH/dihydrofolate, TPNH/ folate, and DPNH/dihydrofolate) when tested near their pH optima. V values increased 3- to 10-fold when TPNH was cofactor; K_m values increased 10- to 15-fold for the TPNH/dihydrofolate pair. The mercurial-activated enzyme, unlike the native form, exhibits a markedly increased sensitivity to heat, proteolysis, and the ionic environment, losing approximately 50% of its activity under conditions where there is no loss of activity in the native form. However, substrates can afford protection, the order of effectiveness being identical with the relative affinities of the substrates for the native enzyme (Subramanian, S., and Kaufman, B. T. (1978) Proc. Nat. Acad. Sci. USA75, 3201). Thus, dihydrofolate, with the largest binding constant is the most efficient, protecting completely against trypsin digestion when present at a 1:1 ratio with enzyme. Heating the mercury enzyme in the absence of substrates gives rise to a stable but altered conformation characterized by a time course which shows marked hysteresis. The striking similarity of the properties of the mercurial-activated dihydrofolate reductase to the reductase activated by 4 m urea, a reagent known to affect the tertiary structure of proteins, suggests that covalent binding of organic mercurials to the sulfhydryl group results in a similar conformational change characterized by a marked facilitation of the dihydrofolate reductase reaction.     

A Comprehensive Genetic Map of Murine Chromosome 11 Reveals Extensive Linkage Conservation between Mouse and Human

Interspecific backcross animals from a cross between C57BL/6J and Mus spretus mice were used to generate a comprehensive linkage map of mouse chromosome 11. The relative map positions of genes previously assigned to mouse chromosome 11 by somatic cell hybrid or genetic backcross analysis were determined (Erbb, Rel, 11-3, Csfgm, Trp53-1, Evi-2, Erba, Erbb-2, Csfg, Myhs, Cola-1, Myla, Hox-2 and Pkca). We also analyzed genes that we suspected would map to chromosome 11 by virtue of their location in human chromosomes and the known linkage homologies that exist between murine chromosome 11 and human chromosomes (Mpo, Ngfr, Pdgfr and Fms). Two of the latter genes, Mpo and Ngfr, mapped to mouse chromosome 11. Both genes also mapped to human chromosome 17, extending the degree of linkage conservation observed between human chromosome 17 and mouse chromosome 11. Pdgfr and Fms, which are closely linked to II-3 and Csfgm in humans on chromosome 5, mapped to mouse chromosome 18 rather than mouse chromosome 11, thereby defining yet another conserved linkage group between human and mouse chromosomes. The mouse chromosome 11 linkage map generated in these studies substantially extends the framework for identifying homologous genes in the mouse that are involved in human disease, for elucidating the genes responsible for several mouse mutations, and for gaining insights into chromosome evolution and genome organization.     

Genetic heterogeneity of gene defects responsible for familial Alzheimer disease

Inherited Alzheimer's disease is a genetically heterogeneous disorder that involves gene defects on at least five chromosomal loci. Three of these loci have been found by genetic linkage studies to reside on chromosomes 21, 19, and 14. On chromosomes 21, the gene encoding the precursor protein of Alzheimerassociated amyloid (APP) has been shown to contain several mutations in exons 16 and 17 which account for roughly 2–3% of familial Alzheimer's disease (FAD). The other loci include what appears to be a susceptibility gene on chromosome 19 associated with late-onset (>65 years) FAD, and a major early-onset FAD gene defect on the long arm of chromosome 14. In other early-and late-onset FAD kindreds, the gene defects involved do not appear to be linked to any of these three loci, indicating the existence of additional and as of yet unlocalized FAD genes. This review provides a historical perspective of the search for FAD gene defects and summarizes the progress made in world-wide attempts to isolate and characterize the genes responsible for this disorder.