6. expressed in mouse liver (25, 26). Recombinant human p.G307S CBS has been produced in several different heterologous expression systems, including lacking the gene was enzymatically active (28). However, the behavior of p.G307S in a mouse model has not been explored. To fully understand the consequences of p.G307S CBS, we have created a mouse (and expresses p.G307S under the control of the zinc-inducible mouse metallothionein (knockout mice XY101 on a C57BL6 strain background show a high frequency of neonatal death, with 90% of the homozygous animals dead by 4 weeks of age due to liver damage (29). Previously, our laboratory has shown that this neonatal lethality can be rescued by the introduction of a transgene expressing either WT or certain mutant human alleles. For this study, we constructed a vector that contains a hemagglutinin-tagged p.G307S encoding cDNA downstream of the mouse promoter flanked by two locus control regions that help alleviate position effect differences in expression. This construct is usually identical (except for the mutation) to our earlier constructs (30). The construct was injected into C57BL6/C3H F2 embryos. From 44 offspring, we obtained eight transgene-positive founders. All of these founders (3, 4, 13, 14, 23, 26, 29, and 36) were then crossed to WT C57BL6 to determine whether the transgene was germline-transmissible. Transgene-positive offspring were obtained from all but one of the lines, and the progeny of four lines (4, 13, 26, and 36) was then assessed for zinc-inducible transgene expression. Immunoblot analysis of the liver indicated that three lines expressed the transgene (13, 26, and 36), whereas one line did not (Fig. 1immunoblot analysis of liver extracts from female mice from the indicated founder line on zinc-water. Blots were probed with a polyclonal serum that recognizes both human (has extract from a control = 13) or Tg-G307S Cbs?/? mice (= 8). shows S.E. immunoblot analysis of p.G307S expression in expression of transgenic CBS under native conditions. Mice from the three expressing lines were then tested to see whether XY101 they could suppress the neonatal lethality phenotype. Suppression of neonatal lethality was observed previously in value? ? value (= 0.0008). These studies confirm that neonatal lethality associated with = 8), which is about a 100-fold increase from that observed in = 4, 0.0001). Mean serum methionine was 95 36 m (= 8), not significantly different from that found in control animals (82 m, = 4, = not significant). At 33C35 days of age (just after weaning), the surviving effect of bortezomib (indicates that the activity was below our limit of detection (20 models). Note that samples in the two different blots with identical tHcy levels are the same. effect of oprozomib (CBS (18,C20, 35,C38). There are a total of nine PDB entries of human CBS (1JBQ, XY101 1M54, 4COO, 4L0D, 4L27, 4L28, 4L3V, 4PCU, and 5MMS) and three PDB entries of CBS (3PC2, 3PC3, and 3PC4). Each structure contains the common dimer found in this family of PLP-dependent Mouse monoclonal to ApoE enzymes (39). The dimers found in XY101 the human structures are shown in Fig. 4in Fig. 4in Fig. 4rotamer (1 dihedral angle 180, in Fig. 4structure 3PC4 contains PLP (in Fig. 4in Fig. 4position also cannot make this interaction (distance 7.4 ?). XY101 Open in a separate window Physique 4. structural superposition of structures of the human CBS homodimer. PLP is usually shown as and respectively. close-up of the active site of CBS. Residues 304C310 are shown as and in (Fig. 5Tyr-308 in the gauche-minus conformation (E-Cyst ligand fits into this volume when Tyr-308 is in the position (rotamer. To explore these possibilities, we performed molecular dynamics simulations of both the WT human CBS and G307S CBS by modeling the serine side chain at position 307 in both the A and B chains of PDB entry 4COO (see Materials and methods). It should be noted that there is a.