␤-Lactamases are bacterial enzymes that hydrolyze ␤-lactam antibiotics to render them inactive. The ␤-lactamase inhibitor protein (BLIP) of Streptomyces clavuligerus, is a potent inhibitor of several ␤-lactamases, including the TEM-1 enzyme (K i ‫؍‬ 0.6 nM). Evidence from the TEM-1/BLIP co-crystal suggests that two BLIP residues, Asp-49 and Phe-142, mimic interactions made by penicillin G when bound in the active site of TEM-1. To determine the importance of these two residues, a heterologous expression system for BLIP was established in Escherichia coli. Site-directed mutagenesis was used to change Asp-49 and Phe-142 to alanine, and inhibition constants (K i ) for both mutants were determined. Each mutation increases the K i for BLIP inhibition of TEM-1 ␤-lactamase approximately 100-fold. To address how these two positions effect the specificity of ␤-lactamase binding, K i values were determined for the interaction of wild-type BLIP, as well as the D49A and F142A mutants, with two extended spectrum ␤-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/␤-lactamase interface. Interestingly, the three BLIP proteins inhibited the G238S ␤-lactamase mutant to the same degree that they inhibited TEM-1. However, wild-type BLIP has a higher K i for the E104K ␤-lactamase mutant, suggesting that interactions between BLIP and ␤-lactamase residue Glu-104 are important for wild-type levels of BLIP inhibition.

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.

␤-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several ␤-lactamases including TEM-1 ␤-lactamase (K i ‫؍‬ 0.1 nM). The co-crystal structure of TEM-1 ␤-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 ␤-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 ␤-lactamase in the 99 -114 loop-helix and 235-240 B3 ␤-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional ␤-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 ␤-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected ␤-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6-and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a K i of 0.40 pM. These results reveal that the binding between TEM-1 ␤-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and ␤-lactamase.

To overcome the antibiotic resistance mechanism mediated by ␤-lactamases, small-molecule ␤-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in ␤-lactamases no longer sensitive to ␤-lactamase inhibitors. On the basis of the structure of ␤-lactamase inhibitor protein (BLIP), novel peptide inhibitors of ␤-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 ␤-lactamase (K i ‫؍‬ 0.3 nM). The cocrystal structure of TEM-1 ␤-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 ␤-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 ␤-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 ␤-lactamase, the peptide also inhibits a class A ␤-lactamase and a class C ␤-lactamase that are not inhibited by BLIP. The crystal structures of class A and C ␤-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar threedimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of ␤-lactamases and PBPs.

Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the lowmolecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis.

DAS-81910-7 cotton is a transgenic event that was transformed to contain the aad-12 and pat genes. These genes code for the AAD-12 and PAT proteins, which confer tolerance to the herbicides 2,4-D and glufosinate, respectively. Crop composition studies were conducted with DAS-81910-7 cotton (both nonsprayed and sprayed with 2,4-D and glufosinate) to comply with requirements of regulatory authorities responsible for evaluating crop safety. Results indicate compositional equivalence between DAS-81910-7 cottonseed and nontransgenic cottonseed and between sprayed and nonsprayed DAS-81910-7 cottonseed. This study builds on the results from many prior studies which support the conclusion that transgenesis is less likely to unexpectedly alter the composition of crops as compared with traditional breeding.

Protein-protein interactions are involved in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.1 nM). To aid in the development of new inhibitors of beta-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 beta-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 beta-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit beta-lactamase.

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