Vector Lambda Research Articles

Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus-infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and titre of anti-S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus-infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and tite of anti-S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinnguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.

Vaccination with a Synthetic Zona Pellucida Peptide Produces Long-Term Contraception in Female Mice

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.

Autogenous regulation of the RNA polymerase beta subunit of Escherichia coli occurs at the translational level in vivo.

A series of transcriptional and translational fusions of the gene for the beta subunit of RNA polymerase (rpoB) to the lacZ reporter gene have been constructed on lambda vectors. Both transcriptional and translational fusions carry the upstream rplKAJL ribosomal protein gene region, which contains the two strong promoters rplKp and rplJp responsible for the transcription of rpoBC. Monolysogens carrying either the transcriptional translational fusion were assayed for beta-galactosidase, providing a measure of the transcription or of both transcription and translation of rpoB, respectively. Translational fusion monolysogens which also carried a multicopy plasmid containing the beta and beta' genes (rpoBC) under the control of a regulatable promoter, exhibited a substantial decrease in the beta-galactosidase levels upon overproduction of beta and beta'. No significant effect was seen in comparable experiments with the transcriptional fusions. These results argue that in vivo, the synthesis of the RNA polymerase beta subunit is autogenously regulated by a translational mechanism. Furthermore, experiments with the overexpressing plasmids confirm the requirement for a portion of the rplL-rpoB intercistronic region in the vicinity of the RNaseIII processing site for the efficient translation of the beta subunit mRNA.

Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

Location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli

The structural gene for deoxyguanosine triphosphate triphosphohydrolase (dGTPase) (EC 3.1.5.1) and its regulator, optA, have been located on a lambda phage carrying a 17.5kb Escherichia coli DNA insert. The DNA fragment has been excised and ligated into pBR325 and also transferred to another lambda vector. From the results of transduction and transformation experiments, we find that the structural gene for dGTPase is very closely linked to optA and dapD, which locates it at approximately 3.6 minutes on the genetic map of E. coli K12. We propose the mnemonic dgt as the designation for the structural gene for this enzyme.

Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein.

A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gt11, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.

Cloning of the bovine major histocompatibility complex class II genes.

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.

Isolation and Characterization of cDNA Clones Encoding Embryonic and Adult Isoforms of Rat Cardiac Troponin T

We have previously reported that troponin T (TnT), a regulatory protein in muscle contraction, undergoes a switch from the larger, acidic embryonic form to the smaller, basic adult form during rat heart development (Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309-7315). To investigate the significance and the molecular mechanism of this isoform switching, cDNA clones encoding rat cardiac TnT were obtained by screening a cDNA library constructed from young rat cardiac poly(A)+ RNAs using the expression vector lambda gt11. Clone RCT10 proved to be a full length clone containing 50 base pairs (bp) of 5'-untranslated sequence, 870 bp of coding sequence, and 196 bp plus poly(A) tail at the 3'-untranslated region. In the other cDNA clone (RCT11), the entire 3'-untranslated sequence and most of the coding sequence were identical with that of RCT10, but an additional 30-bp insert was present in the coding region from residues 18 to 27. This insertion sequence appeared to code for a fragment (EDWSEEEEDE) highly enriched in acidic amino acid residues. Thus, RCT11 might represent a clone encoding the embryonic isoform of rat cardiac TnT. S1 nuclease RNA mapping analysis using end-labeled RCT11 cDNA probes confirmed that this region of the sequence is different in embryonic and adult isoform mRNAs. These results suggest that both embryonic and adult isoforms of rat cardiac TnT are generated from the same primary transcript by developmentally regulated alternative splicing. The amino acid sequence deduced from RCT10 cDNA exhibits 87%, 85%, and 72% homology with bovine, rabbit, and chicken cardiac TnTs, respectively, but less homology (57-59%) with the known skeletal TnTs from human, rat, rabbit, and chicken. Moreover, both the 5'- and the 3'-untranslated sequences of rat cardiac TnT mRNA are completely different from those reported for rat skeletal TnT mRNA, suggesting that rat cardiac TnT is coded from a gene distinct from the rat skeletal TnT gene.

A monoclonal antibody to von Willebrand factor (vWF) inhibits factor VIII binding. Localization of its antigenic determinant to a nonadecapeptide at the amino terminus of the mature vWF polypeptide.

vWF is a multimeric glycoprotein that serves as the major carrier in plasma of Factor VIII (FVIII). We have used an anti-human vWF MAb W5-6A to investigate the FVIII binding site on vWF. W5-6A inhibited FVIII binding to vWF-coated polystyrene tubes in a concentration-dependent manner with 90% inhibition of FVIII binding at a concentration of 10 micrograms/ml. The W5-6A epitope was identified by screening a vWF fragment library using the bacteriophage expression vector lambda gt11. DNA sequence analysis of 29 immunoreactive phage clones localized the W5-6A epitope to a nonadecapeptide spanning amino acid residues threonine 78 to threonine 96 at the amino-terminus of the mature vWF polypeptide. Purified beta-galactosidase/vWF fusion protein from one of these clones, vWF9, was incubated with radiolabeled W5-6A and caused near complete inhibition of W5-6A binding to vWF. Inhibitory activity was lost after vWF9 trypsinization or reduction and alkylation. These data indicate that (a) the antigenic determinant recognized by W5-6A localizes to a nonadecapeptide at the NH2 terminus of the mature vWF polypeptide, (b) disulfide bonds within vWF9 may be necessary to maintain the structure required for immunoreactivity with W5-6A, and (c) W5-6A recognizes an immunogenic region on vWF that may be at (or near) the major FVIII binding domain.

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli.

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.

Cloning and sequencing of the genes of 2-hydoxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the lambda vectors gt11 and EMBL 3 were screened with antisera raised against 2-hydroxyglutaryl-CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a lambda gt11 clone with an insert of 2050 bp and one was a lambda EMBL-3 clone with an insert of approximately 11,000 bp. Escherichia coli cells infected with the lambda gt11 clone expressed the alpha subunit of the dehydratase (Mr, 53,870), whereas with the lambda EMBL-3 clone, the alpha and beta subunits (Mr, 41,857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (alpha) and hgdB (beta) were obtained. The genes have the following order: A-B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the alpha and beta subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in alpha, including a CNC and two CC clusters, and nine in beta) but no similarities to other known protein sequences were found.

Isolation and structural characterization of cDNA clones encoding the mating pheromone Er-1 secreted by the ciliate Euplotes raikovi.

cDNA clones comprising the entire coding region for the mating pheromone Er-1 of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors lambda gt10 and pUC12. The cDNA sequence contains an open reading frame of 75 amino acids that constitute pre-pro-Er-1. The amino acid sequence of secreted Er-1 starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-1 protein sequencing. The coding region of Er-1 cDNA ends with codon TAA, which specifies glutamine in other ciliates. The 5'- and 3'-noncoding regions contain, respectively, two and one inverted repeats. The 3'-noncoding-region inverted repeat, which includes the unusual polyadenylylation signal AACAAA, has been related to RNA 3'-terminus formation.

Structural homology of complement protein C6 with other channel-forming proteins of complement.

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.

Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification.

The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.

Construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent vector

We have designed a simple procedure for the construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent cloning vector. An oligonucleotide, its 5' end starting with a heteropolymeric sequence encoding the rare restriction sites for NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand synthesis and EcoRI linker addition, the cDNA is double digested with EcoRI and NotI, or with EcoRI and SfiI, to generate DNA fragments with asymmetric ends that can be directionally cloned. The cDNA fragments are enriched for "full length" by size selection and ligated into a phage lambda vector containing the T3 and T7 RNA polymerase promoters. These cDNA libraries can directly be used for in vitro synthesis of sense or antisense RNA.

Monoclonal antibodies used to characterize cDNA clones expressing specific wheat endosperm proteins

A wheat cDNA library, prepared from grain endosperm poly A + -mRNA and cloned into the Escherichia coli expression vector lambda gt11, has been screened with nine monoclonal antibodies having specificities for different wheat endosperm proteins. At least one positive cDNA clone was isolated, and purified, from those selected with each antibody. Each purified cDNA clone was induced to express fusion proteins, and the nitrocellulose membranes to which the proteins were transferred were incubated with each of the other antibodies at two or more concentrations to investigate the extent of homologies between expressed fusion proteins. The specificities of the antibodies were determined using immunoblotting under the same conditions used for binding to the fusion proteins from the expressed cDNA clones. Denatured DNA from each antibody-selected cDNA clone was also characterized by hybridization to αa-/β-genomic gliadin and genomic high molecular weight glutenin subunit DNA probes. Northern hybridizations using the isolated cDNAs as probes for endosperm mRNA were also used to assist clone identification. Some monoclonal antibodies with overlapping specificities (identified from blotting and ELISA experiments) cross-reacted with several expressed cDNA clones. However, in other instances, positive identifications were made of the proteins coded for by single families of the cDNA clones. Monoclonal antibody screening of a wheat cDNA library is useful in identifying families of cDNA clones corresponding to different wheat polypeptides at the primary screening stage, obviating the need in many instances for the application of more tedious methods of clone identification.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene

By use of anti-cytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kilobases was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a 5'-noncoding region of 25 nucleotides, the coding region of 1,521 nucleotides, and a 3'-nontranslated region of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507-amino acid residues (predicted Mr = 54,462), which contained an additional sequence of 41-amino acid residues on the NH2 terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides produced by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides obtained from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern and Southern blot, and primer-extension analyses using appropriate cDNA segments prepared with fumarase cDNA (1.7 kilobases) as a probe or primer suggested that the mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene and a possibility that the precursors of both these fumarases were synthesized by one species of mRNA having a base sequence coding the presequence of the mitochondrial fumarase by some unknown post-transcriptional mechanism(s).