Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops.

Diverse Fab libraries containing 2-3 x 10(8) members were generated by randomizing amino acid residues within four of the six complementarity determining regions of a humanized version of an anti-HER-2 Ab (hu4D5). These libraries were subsequently displayed on the surface of the filamentous bacteriophage M13 and selected for binding to three proteins: CD4, insulin-like growth factor 1 (IGF-1), and tissue plasminogen activator. An Fab-bacteriophage was isolated that showed specific binding to IGF-1. The affinity of this Fab was determined to be 3.5 microM.

Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli.

A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E. coli rph gene, which was formerly called orfE. This B. subtilis gene is located at 251 degrees adjacent to the gerM gene on the B. subtilis genetic map. It has been named rph because, like its E. coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.

Fab assembly and enrichment in a monovalent phage display system.

We have developed a system that allows the expression of a single copy of an antibody Fab molecule on the surface of the filamentous bacteriophage M13 and demonstrate the utility of this system for enrichment of specific "Fab phage". A "humanized" version of antibody 4D5 (h4D5) directed against the extracellular domain of the HER2 (neu) receptor, was used as prototype to assess the assembly of Fab molecules on the phage and to determine the power of the enrichment system. The h4D5 Fab phage showed specific binding to the extracellular domain of the receptor and exhibited an affinity for its antigen virtually identical to the Fab itself. By virtue of its antigen binding, the h4D5 Fab phage could be sorted from a million-fold excess of non-cognate Fab phage in only two rounds of sorting. Further experiments demonstrated that the h4D5 Fab phage could be preferentially enriched from mixtures of variant Fab phages that had lower affinities for the HER2 receptor.

An SfiI restriction map of the Bacillus subtilis 168 genome.

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.

High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis.

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.

The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis.

We have cloned and characterized the mtr operon of Bacillus subtilis. This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon. We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively. MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4. The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter. Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein. The in vivo action of the mtrA and mtrB gene products was analyzed in an E. coli strain containing a trpE-lacZ gene fusion under control of the B. subtilis trp promoter/attenuator region. Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.

The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Engineering human prolactin to bind to the human growth hormone receptor.

A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL). The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver. After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold. This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH. These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.

Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems.

Mutations in the sacU region have a pleiotropic phenotype. Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile. We isolated and sequenced the sacU gene region of Bacillus subtilis. Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames. The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli.

Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene.

The immediate-early IE-1 gene of Autographa californica nuclear polyhedrosis virus was cloned, and its nucleotide sequence was determined. Sequence analysis indicated that this gene would encode a protein of 582 amino acids with a predicted molecular weight of 66,822. Analysis of IE-1 gene expression during baculovirus infection identified two transcripts. One, 1.9 kilobases (kb), was expressed at constant steady-state levels throughout infection, whereas the other, 2.1 kb, was expressed only early in infection. Analysis of IE-1 cDNA clones demonstrated that the 2.1-kb transcript contained the entire 1.9-kb transcript (exon 1) plus an additional 5' end (exon 0). Genomic Southern analysis placed the exon 0 sequences on the EcoRI B fragment, 4 kilobase pairs upstream of exon 1. Sequencing of the upstream region identified an open reading frame whose 5' end was identical to the exon 0 sequences in the cDNAs. Examination of the genomic DNA sequences around the exon-exon junction revealed sequences similar to published consensus splice acceptor and donor sequences. This is the first example of splicing of any viral transcript during baculovirus infection.

Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants.

The start point for transcription of the subtilisin (aprE) gene was determined by primer extension analysis and was found to be at a point significantly different from that identified in a previously published report (S. L. Wong, C. W. Price, D. S. Goldfarb, and R. H. Doi, Proc. Natl. Acad. Sci. USA 81:1184-1188, 1984). An aprE-lacZ fusion was used to analyze expression of the promoter. Deletion analyses of the promoter were performed to determine the extent of the upstream region necessary for activity. This was found to be between -52 and -41 with respect to the transcription start site. Expression of the aprE-lacZ fusion was unimpaired in a mutant deleted for the sigma B subunit of RNA polymerase. Mutations in the gene for the sigma H subunit of RNA polymerase decreased expression of the aprE-lacZ fusion to approximately 25% of that of the wild type. These results leave the identity of the sigma factor responsible for transcription of this gene in question. Mutations in the spo0A gene drastically decreased the activity of the aprE promoter and its upstream deletion derivatives, while the abrB gene, a phenotypic suppressor of spo0 mutations, restored activity of the aprE promoter in all of the deletion derivatives. Thus, inhibition of transcription by the spo0A mutation and its restoration by an abrB mutation could not be separated from the promoter of the aprE gene.

Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter.

A number of mutations have been described with pleiotropic effects on the expression of genes for degradative enzymes in Bacillus subtilis. The sacU32(Hy) and sacQ36(Hy) mutations increase the expression of a wide variety of enzymes that degrade biological polymers. The phenotypes caused by mutations at the hpr locus are more restricted; they are known to increase expression of the alkaline and neutral proteases. The alkaline protease (aprE) promoter was analyzed to determine the target site for stimulation by these loci. Deletion of upstream regions of the aprE promoter could abolish or greatly reduce stimulation by mutations at these loci. A region upstream of -200 was necessary for full stimulation by an hpr-97 mutation, whereas a region between -141 and -164 was necessary for full stimulation by the sacU32(Hy) and sacQ36(Hy) mutations. Northern analyses of mRNA preparations showed that the levels of aprE mRNA were increased in strains carrying the sacU32(Hy) or hpr-97 mutation. Moreover, primer extension analysis of these mRNA preparations revealed that the transcription start point was identical to that in a wild-type strain. We hypothesize that upstream activation of the subtilisin promoter mediated by these genes is a mechanism for global responses to a variety of nutritional conditions.

Sequence and centromere proximal location of a transformation enhancing fragment ans1 from Aspergillus nidulans.

The Aspergillus nidulans sequence ans1, previously known to enhance transformation frequencies of pyr4-based vectors, was shown to enhance the efficiency of argB and trpC-based vectors. Increased efficiencies could be obtained by constructing vectors containing argB and ans1 or by cotransforming selectable plasmids (containing argB, trpC, or pyr4) with the non-selectable ans1 sequence. The preponderance of evidence suggests that the mechanism of ans1 activity does not involve homologous recombination events, in spite of the presence of multiple regions of homology in the A. nidulans genome. Genetic mapping localized ans1 to the vicinity of the centromere of linkage group I. The nucleotide sequence of a 1.8 Kb functional subclone of ans1 was determined and found to be highly A + T rich (81%).

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph.

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.

Characterization and mapping of the Bacillus subtilis prtR gene.

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.