Three DNA markers for hypophosphataemic rickets

Summary This paper presents three markers, 16D/E, pHMAI (DXS208), and CRI-L1391 (DXS274), that show close linkage for X-linked hypophosphataemic rickets (HYP). DXS274 is closely linked to HYP ( _max= 0.00, Z_max = 4.20), and DXS41 (99.6), ( _max= 0.00, Z_max = 5.20). Marker 16D/E maps distal to the disease locus ( _max= 0.05, Z_max = 3.11). The pHMAI probe recognises the same restriction fragment length polymorphism (RFLP) as 99.6. Multipoint analysis suggests that the most probable order of loci is Xpter-(DXS43, 16D/E)-HYP-DXS274-(DXS208, DXS41)-Xcen. The location of DXS274 distal to HYP cannot be excluded, as no recombinants were observed between DXS274 and HYP, or between DXS274 and DXS41/DXS208. One of the families contains a large number of recombinants, four of which are double recombinants. This most probably means that the disease in this family maps elsewhere on the X chromosome or on an autosome, indicating locus heterogeneity.     

Sequential determination of ligands binding to discrete components in heterogeneous mixtures by iterative panning and blocking (IPAB)

Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.     

The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha

The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397). Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.