Distribution of Cryptosporidium parvum gp60 subtypes in calf herds of Saxony, Germany.

Cryptosporidiosis is a common protozoan parasitic infection that causes diarrhoea in neonatal calves. The high shedding of environmentally resistant oocysts facilitates outbreaks of cryptosporidiosis in humans. In total, 58 farms (512 calves) in Germany (Saxony and Brandenburg) were visited three times each. Faecal samples of pre-weaned calves were microscopically examined for oocysts of Cryptosporidium spp. using Heine staining and were scored with regard to their consistency. Overall, 88.9% of calves tested microscopically positive for Cryptosporidium spp. in at least one sample, and the excretion of oocysts was significantly (P < 0.01) associated with a higher faecal score (diarrhoea). After DNA extraction from pooled farm isolates, 47 samples were successfully subtyped by sequence analysis of the 60 kDa glycoprotein gene (gp60). All isolates belonged to subtype family IIa. IIaA15G2R1 was the most common subtype (present on 66% of the farms), followed by IIaA16G3R1 (13%). Subtypes IIaA14G1R1, IIaA14G2R1, IIaA1612R1, IIaA16G2R1, IIaA17G1R1, IIaA17G2R1, IIaA17G4R1 and IIaA19G2R1 were found sporadically. This is the first description of gp60 subtype IIaA17G4R1 in cattle in Germany.

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

The scaffold/matrix attachment region binding protein hnRNP-U (SAF-A) is directly bound to chromosomal DNA in vivo: a chemical cross-linking study.

The protein heterogeneous nuclear ribonucleoprotein U (hnRNP-U, also known as scaffold attachment factor A, SAF-A) is an abundant component of hnRNP particles and of the nuclear matrix. Previous experiments have demonstrated that, in vitro, hnRNP-U specifically binds to scaffold/matrix attachment (S/MAR) region DNA elements and could thus be involved in higher order chromatin structure. In this paper we report on the use of chemical cross-linking to investigate whether the protein is also bound to DNA in vivo, which is a prerequisite for its presumed function in chromatin loop formation. We have improved published methods for cross-linking proteins to DNA with the aim to minimize unspecific fixation and possible contamination with RNA binding proteins. Our protocol is based on a limited cross-linking of living human cells with formaldehyde, followed by the purification of DNA/protein complexes by two consecutive cesium chloride density gradient centrifugations. Analysis of the protein constituents of these complexes shows a specific subset of cross-linked proteins with the histones as major components. By western blotting, we demonstrate that hnRNP-U is efficiently cross-linked to DNA under experimental conditions that yield DNA/protein complexes with a buoyant density equivalent to that of native chromatin. Dimethylsulfate cross-linking and limited protease digestion of the complexes was used to establish that hnRNP-U is bound directly to DNA and not via cross-linking to other proteins. This is the first direct demonstration of the in vivo DNA binding of a S/MAR specific protein and suggests a structural role of hnRNP-U in chromatin organization.

The novel SAR-binding domain of scaffold attachment factor A (SAF-A) is a target in apoptotic nuclear breakdown.

The scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Evidence from previous experiments had suggested that SAF-A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism. We now show that the protein has a novel scaffold-associated region (SAR)-specific bipartite DNA-binding domain which is independent from the previously identified RNA-binding domain, the RGG box. During apoptosis, but not during necrosis, SAF-A is cleaved in a caspase-dependent way. Cleavage occurs within the bipartite DNA-binding domain, resulting in a loss of DNA-binding activity and a concomitant detachment of SAF-A from nuclear structural sites. On the other hand, cleavage does not compromise the association of SAF-A with hnRNP complexes, indicating that the function of SAF-A in RNA metabolism is not affected in apoptosis. Our results suggest that detachment of SAF-A from SARs, caused by apoptotic proteolysis of its DNA-binding domain, is linked to the formation of oligonucleosomal-sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells.

Interactions of human nuclear proteins P1Mcm3 and P1Cdc46.

Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication. Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin. At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46. However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm. About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46. These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa). The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers. It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication. Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer. DNA polymerase alpha is not a component of this complex.