Start Date: Jul 24, 2007
End Date: Jul 23, 2012
Objective 1: PCR cloning will be utilized to fuse the mature form of lysostaphin to the phi11 endolysin and to the LysK endolysin. These fusion constructs will have CHAP endopeptidase, amidase and glycyl-glycyl endopeptidase domains. Imidazole step gradient on a nickel column will yield a phi11 endolysin-lysostaphin that is free of contaminating protein, to be used against S. aureus in a turbidity assay. A similar imidazole step gradient will be used to purify the LysK endolysin-lysostaphin construct. To determine if the endolysins (CHAP or amidase) contribute to cell lysis, each domain will be subjected to site-directed mutagenesis to inactivate domains one at a time. Objective 2. Tissues will be collected at slaughter from lactating transgenic females and pregnant females from each of the three lines, and from non-transgenic lactating females (negative control). Tissues will also be collected from at least one sexually mature bull from each of the lines. The tissues will be stored at -80C or 0.5 gm will be added to 2.5 ml of RNAlater, minced and then frozen until processed. The tissues to be collected include brain, blood, gonads, heart, kidney, muscle, liver, lung, lymph node, salivary gland, and spleen. All samples will be assayed for lysostaphin transgene mRNA expression (normalized to beta-actin expression), protein and biological activity as we have previously described. For gene-specific DNA methylation and histone acetylation, the bisulfite sequencing and Chromatin precipitation assay (CHIP) will be used. The methylation of cytosines in CpG dinucleotides will be determined from the sequence information of at least 10 independent clones. For the CHIP assay, antibodies specific to acetylated histones will be incubated with DNA in cells after fixation. Amplification of specific genes will be conducted by real-time PCR after bound DNA was precipitated by centrifugation. The amount of histone acetylation can be determined by the levels of amplification. Synaptonemal complex structure and chiasmata localization will be characterized by immunofluorescent staining of SCP3 (synaptonemal complex lateral element protein 3) and MLH1 (MutL homolog 1 mismatch repair protein) foci.