Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

X-ray crystallographic studies of myoglobin do not show an entrance or exit path for potential ligands from the surface to the heme cavity. Efforts to locate such a path have so far centered around dynamic calculations. A structure has now been determined that has a clear opening. Phenylhydrazine reacts with myoglobin in such a way that a phenyl group remains bound to the iron atom. The structure of this complex shows that the side chains of His-64(E7), Arg-45-(CD3), and Val-68(E11) have been forced aside to form an open channel to the surface. Although this may not be the only channel to the iron atom, it seems likely that it is an important one.

Histone N-terminal domains are frequent targets of posttranslational modifications. Multiple acetylated lysine residues have been identified in the N-terminal domain of H2B (K6, K11, K16, K17, K21, and K22), but little is known about how these modifications regulate transcription. We systematically mutated the N-terminal domain of histone H2B, both at known sites of lysine acetylation and elsewhere, and characterized the resulting changes in genome-wide expression in each mutant strain. Our results indicate that known sites of lysine acetylation in this domain are required for gene-specific transcriptional activation. However, the entire H2B N-terminal domain is principally required for the transcriptional repression of a large subset of the yeast genome. We find that the histone H2B repression (HBR) domain, comprised of residues 30 to 37, is necessary and sufficient for this repression. Many of the genes repressed by the HBR domain are located adjacent to telomeres or function in vitamin and carbohydrate metabolism. Deletion of the HBR domain also confers an increased sensitivity to DNA damage by UV irradiation. We mapped the critical residues in the HBR domain required for its repression function. Finally, comparisons of these data with previous studies reveal that a surprising number of genes are coregulated by the N-terminal domains of histone H2B, H3, and H4.In eukaryotic cells, DNA is packaged with histones and other proteins into chromatin (5). The principal packaging unit is the nucleosome, which consists of approximately 147 bp of DNA wrapped around a protein core of one histone H3-H4 tetramer and two histone H2A-H2B dimers (21). Because of their close association with DNA in the nucleosome, histones play integral roles in DNA-templated processes, such as DNA transcription, replication, and repair. Each of the four histone proteins is covalently modified at multiple residues, principally in their flexible N-terminal domains (9, 15). Histone modifications, such as lysine acetylation, lysine methylation, and serine phosphorylation, profoundly affect transcription initiation by regulating the association of transcriptional regulatory proteins with DNA (2, 10, 30).While modifications in the N-terminal domains of histone H3 and H4 have been extensively studied, relatively little is known about how N-terminal modifications in histone H2A and H2B regulate transcription. In Saccharomyces cerevisiae, histone H2B is acetylated at six lysine residues in its N-terminal domain (K6, K11, K16, K17, K21, and K22), most likely by the Gcn5 histone acetyltransferase (24, 28). H2B-K16 has been shown to be hypoacetylated in regions of subtelomeric heterochromatin due to the actions of the Hda1 histone deacetylase (23). It is unclear, however, whether there is a functional requirement for histone H2B hypoacetylation in subtelomeric heterochromatin. The histone H2B N-terminal domain does not appear to regulate telomeric silencing (29, 32).Deletion of amino acids 30 to 37 in the H2B N-terminal domain, which is lethal in some s...

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Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.