A rapid, flexible method for incorporating controlled antibiotic release into porous polymethylmethacrylate space maintainers for craniofacial reconstruction.

Severe injuries in the craniofacial complex, resulting from trauma or pathology, present several challenges to functional and aesthetic reconstruction. The anatomy and position of the craniofacial region make it vulnerable to injury and subsequent local infection due to external bacteria as well as those from neighbouring structures like the sinuses, nasal passages, and mouth. Porous polymethylmethacrylate (PMMA) "space maintainers" have proven useful in staged craniofacial reconstruction by promoting healing of overlying soft tissue prior to reconstruction of craniofacial bones. We describe herein a method by which the porosity of a prefabricated porous PMMA space maintainer, generated by porogen leaching, can be loaded with a thermogelling copolymer-based drug delivery system. Porogen leaching, space maintainer prewetting, and thermogel loading all significantly affected the loading of a model antibiotic, colistin. Weeks-long release of antibiotic at clinically relevant levels was achieved with several formulations. In vitro assays confirmed that the released colistin maintained its antibiotic activity against several bacterial targets. Our results suggest that this method is a valuable tool in the development of novel therapeutic approaches for the treatment of severe complex, infected craniofacial injuries.

Enzymatic digestion of operator DNA in the presence of the lac repressor tryptic core.

The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3' boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3' boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene.

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.

A method for construction of E. coli strains with multiple DNA insertions in the chromosome.

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage lambda, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn+ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage lambda xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the lambda attL and lambda attR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.

Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator.

A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites. Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids. The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests. The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence. Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site. These derivatives showed in vivo operator activity. Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed. These plasmids should be useful in preparing large amounts of the 41-bp fragment.

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. II. Insertion of lac operator fragments.

To examine the effect of introducing protein-binding sites around a promoter upon expression from that promoter, a series of altered tryptophan promoter plasmids have been constructed. In these derivatives of pKO-1 (a galK-expression, promoter-assay vector), the trp promoter has been introduced into the vector, such that the galK expression is dependent on the trp promoter. Unique restriction sites have been introduced adjacent to the trp promoter for the insertion of other DNA fragments. The DNA-binding site (a lac operator (lacO) fragment of 33 bp) for the lactose repressor was inserted into the trp promoter-pKO plasmids at various defined locations from -52 to +58 relative to the start site of transcription. Strains bearing tryptophan promoter-lactose operator plasmid derivatives were assayed for galactokinase activity in Escherichia coli C600 and JM103 (a lacIQ strain) to observe the effect of lac repressor binding and removal. In those plasmids where the lac repressor was downstream from the promoter, expression was diminished by the presence of the lactose repressor and galactokinase activity could be induced by addition of IPTG. An analogous lac promoter plasmid was repressed over 90%, and the plasmid containing the lacO fragment at +2 exhibited up to 80% repression; 40-50% repression was observed when the lacO was placed at positions +27 and +58. Placement of the lacO at the upstream locations -39 and -52 produced lower repression. To examine the effects of more than one operator surrounding the promoter, plasmids were constructed that had a lacO at -39 or -52 and, in addition, had an operator downstream.+2

Sequence and arrangement of genes encoding sigma factors in Clostridium acetobutylicum ATCC 824.

The nucleotide sequence of a 2.7-kb region of Clostridium acetobutylicum ATCC 824 DNA containing three open reading frames was determined. They encoded homologs of three proteins of Bacillus subtilis, and the gene arrangement in both organisms was identical. The first gene, orfA, was 801-bp long; the 31-kDa (266 aa) product it encoded exhibited homology with the putative sigma E-processing enzyme. The second gene, sigE, was 708-bp long encoding a 27-kDa (235 aa) product; the third gene, sigG, was 774-bp long encoding a 30-kDa (257 aa) product. These two proteins showed high homology with sigma E and sigma G, two sporulation-specific sigma factors.

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Use of bacterial DHFR-II fusion proteins to elicit specific antibodies.

Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.

Characterization of an acetyl-CoA C-acetyltransferase (thiolase) gene from Clostridium acetobutylicum ATCC 824.

Thiolase (Thl) is an important enzyme at the junction in the pathway leading to the production of either acids (acetate or butyrate) or solvents (acetone, butanol or ethanol) during the growth of Clostridium acetobutylicum ATCC 824. Cloning and expression of the Thl-encoding gene (thl) has been described [Petersen and Bennett, Appl. Environ. Microbiol. 57 (1991) 2735-2741], as has the purification and properties of the enzyme [Wiesenborn et al., Appl. Environ. Microbiol. 54 (1988) 2717-2722]. Here, we present the complete nucleotide sequence (1.9 kb) of thl. The gene encodes a protein of 392 amino acids (aa) (41,237 Da), which mass is in agreement with previous findings using the purified protein. Primer extension analysis has defined the promoter region, and a stem-loop structure found at the end of thl indicates that it is not part of an operon. The aa sequence of Thl showed homology to those of four other beta-ketothiolases: (i) PhbC of Alcaligenes eutrophus, (ii) PhbA of Chromatium vinosum, (iii) PhbA of Thiocystis violacea and (iv) PhbA of Zoogloea ramigera. The C terminus of an open reading frame found upstream from the Thl sequence is similar to OrfX of Bacillus subtilis and to NfrC of Escherichia coli.

Sequence and arrangement of genes encoding enzymes of the acetone-production pathway of Clostridium acetobutylicum ATCC824.

The nucleotide sequence of three open reading frames in the acetone-production locus of Clostridium acetobutylicum ATCC824 has been established. The three gene products, corresponding to acetoacetate decarboxylase (EC 4.1.1.4) and both subunits of the acetoacetyl-CoA:acetate/butyrate:CoA transferase (EC 2.8.3.9) are transcribed in two convergently arranged operons. The intervening DNA region separating the two transcripts is characterized by an inverted repeat which appears capable of forming a stem-loop structure functioning as a Rho-independent transcription terminator in both directions.

Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824.

The genes encoding both Clostridium acetobutylicum ATCC 824 butyrate synthesis pathway enzymes, phosphotransbutyrylase (ptb) and butyrate kinase (buk), were sequenced. The genes are immediately adjacent on the chromosome, with ptb preceding buk. A single transcription start point (tsp) was identified 57 bp upstream from the ptb start codon by primer extension analysis. The ptb and buk genes appear to form an operon. A putative Rho-independent terminator structure was identified 26 bp downstream from buk.

Formation of MboII vectors and cassettes using asymmetric MboII linkers.

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.

Escherichia coli strain for thermoinducible T7 RNA polymerase-driven expression.

An Escherichia coli strain for thermoinducible T7Pol-driven transcription has been constructed. The strain was obtained by site-specific integration of the T7 gene 1 coding for T7Pol into the attB site of phage lambda in the E. coli chromosome. The expression of the inserted gene is regulated by cIts857 and major early promoter-operator regions of phage lambda.

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides.

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.

Cloning and assembly of PCR products using modified primers and DNA repair enzymes.

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.

Methods for cloning key primary metabolic enzymes and ancillary proteins associated with the acetone-butanol fermentation of Clostridium acetobutylicum.

The unavailability of genetically defined mutants for complementation has intensified the problems inherent in cloning genes from C. acetobutylicum. The uniqueness of some of the pathways of this organism coupled with the relative inefficiency of transformation of clostridia and few characterized mutants in these pathways have made cloning these genes by traditional complementation methods impractical. Oligonucleotide hybridization techniques have been shown to circumvent many problems involved in detecting protein expression. The ease of hybridization screening of plaques allows phage libraries to be examined more readily than is generally the case with colony screening techniques. Recombinant lambda phages also contain more DNA per insert than most plasmid vectors can maintain, thus further decreasing the amount of screening necessary. Cosmid libraries, offering even greater length of individual inserts, can be screened in a similar manner, although such screening incorporates the limitations of colony screening techniques. It is true that the technique hinges on the ability to obtain an amino acid sequence from which an oligonucleotide can be designed. In the past, the ability to obtain sequences was limited because the quantity and number of purified proteins were limited or the proteins were amino-terminally blocked. However, recent technological advances in this area, such as high-resolution gel separation techniques coupled with microsequencing, have opened the door to proteins previously inaccessible. Deformylation methods have been developed to deblock amino-terminally formylated proteins, and successful internal amino acid sequence analysis by in situ protease digestion has also been reported using only picomolar quantities of proteins separated by one- or two-dimensional gel electrophoresis. Protein and DNA sequence data banks have been significantly upgraded in the past few years. A proposed oligonucleotide sequence can be evaluated to determine what other possible sequences have similar homology; moreover, protein similarity comparisons between related species might possibly supplant the need for protein isolation if regions of highly conserved amino acid sequences are found. To our knowledge, this represents the first reported use of oligonucleotide probe hybridization screening technology as a strategy for cloning solvent pathway genes of C. acetobutylicum. Despite the deleterious effects on hybridization inherent in the high A + T content of C. acetobutylicum gene specific-directed oligonucleotides, the technique has been shown to function with few modifications to previously recorded systems.

Strategies in high-level expression of recombinant protein in Escherichia coli.

The accumulation of acetate is one of the most commonly encountered problems in attaining high levels of recombinant protein production using E. coli. Two different approaches are examined to reduce the rate of acetate formation. The effects of reduced acetate accumulation on recombinant protein production were also investigated. In the first approach, E. coli mutant strains deficient in enzymes involved in the acetate synthesis pathways were isolated and characterized. The level of specific production of beta-galactosidase by the mutant strain is three times higher than its parent strain. In another approach, metabolic engineering techniques were employed to fine-tune the central metabolic pathways to reduce the amount of acetate formation. The resulting strain, which carries the acetolactase synthase gene from B. subtilis, is successful in maintaining a very low level of acetate accumulation. The ALS-containing strain is also capable of producing higher levels of recombinant protein than its parent strain.

An optimization study of a pH-inducible promoter system for high-level recombinant protein production in Escherichia coli.

The unique properties exhibited by the pH-inducible promoter system are clearly demonstrated by the plasmid construct, pSM552-545C-. Step changes of pH substantially increase the expression of beta-galactosidase. Very high expression, a level of around 40% of total cellular protein, can be achieved with superbroth. The high level of induction in rich media, typical of those commonly used to achieve high cell density, suggests the system is versatile enough to be adapted to many specific situations. The variable degree of induction by pH within the range of 8.0 and 5.5 makes possible a degree of expression control not easily accomplished with the existing systems. By precise monitoring of induction pH, a "fine tuning" of foreign gene expression and growth rate to optimum levels is possible. The effect of several operating parameters on recombinant protein production are evaluated. Our results show that operating environments play an extremely important role in achieving high recombinant protein expression levels in a dense culture. Under suboptimal conditions, as are shown in this study, only moderately high levels can be obtained. Even for suboptimal cases, an expression level of about 10 to 15% of total cellular protein while achieving an optical density higher than 25 is routinely obtained. Our results also show that a proper balance between cell growth and recombinant protein synthesis processes are critical in maintaining high expression levels in a dense culture. Any imbalance will most likely lead to more cell growth and poorer protein productivity. We have also demonstrated that reactor operating temperature can be a useful parameter to fine-tune this balance, resulting in significantly improved results.