The muc genes of pKM101 are induced by DNA damage

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.     

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.     

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.     

Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus-infected humans.

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide. A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of beta-galactosidase was expressed and characterized in bacterial extracts. Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera. The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X. As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.     

Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen.

The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization. A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera. These sera contained high titres of antibodies to a fusion protein, produced in E. coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4. Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD. The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.     

Overlapping domains of the heterochromatin-associated protein HP1 mediate nuclear localization and heterochromatin binding

The Drosophila protein HP1 is a 206 amino acid heterochromatin- associated nonhistone chromosomal protein. Based on the characterization of HP1 to date, there are three properties intrinsic to HP1: nuclear localization, heterochromatin binding, and gene silencing. In this work, we have concentrated on the identification of domains responsible for the nuclear localization and heterochromatin binding properties of HP1. We have expressed a series of beta- galactosidase/HP1 fusion proteins in Drosophila embryos and polytene tissue and have used beta-galactosidase enzymatic activity to identify the subcellular localization of each fusion protein. We have identified two functional domains in HP1: a nuclear localization domain of amino acids 152-206 and a heterochromatin binding domain of amino acids 95- 206. Both of these functional domains overlap an evolutionarily conserved COOH-terminal region.     

Characterization and regulation of a second gene encoding thioredoxin from the fission yeast

A genomic DNA encoding a second thioredoxin (TRX2) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The cloned sequence contains 1823 bp and encodes a protein of 121 amino acids. It has extra N-terminal 17 amino acid residues compared to previously identified thioredoxin (TRX1), which are positively charged and hydrophobic amino acids. The additional N-terminal region contains a plausible prepeptidase cleavage site, indicating that the TRX2 protein exists in mitochondria. The cloned TRX2 gene produced functional TRX estimated with insulin reduction assay. The upstream region of the TRX2 gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R. The 782 bp sequence in the region further upstream of the TRX2 gene was found to be inhibitory in its expression. Synthesis of beta-galactosidase from the fusion plasmid pYFX135-HRL was enhanced by the addition of aluminum chloride and ferrous chloride, indicating that the TRX2 protein is involved in stress response.     

Expression of functional beta-galactosidase containing the coxsackievirus 3C protease as an internal fusion

Alpha complementation of beta-galactosidase (beta gal) is intracistronic and requires interaction between the alpha donor region (residues 3-41) and alpha acceptor fragment (produced by M15). We have constructed two plasmids which direct the synthesis of hybrid beta gal: coxsackievirus proteins in Escherichia coli. One plasmid, pBD1045, encodes an enzymatically active 3C protease of coxsackievirus B3 fused between the amino-terminal 79 amino acids of beta gal (containing the alpha donor region) and amino acids 80 to 1023 (alpha acceptor region). A second plasmid, pBD1043 encodes an inactive 3C protease and results in a fusion of 260 coxsackievirus amino acids between residues 79 and 80 of the beta gal monomer. Both hybrid proteins expressed by these constructs have beta-galactosidase activity regardless of whether the viral protease (183 amino acids) is autocatalytically cleaved out of the chimeric protein (pBD1045) or remains as part of a fusion protein (pBD1043). The implications of these results for structural flexibility of the complemented beta-galactosidase enzyme are discussed.     

Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies.

We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein. Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies. The cDNA fragment is 601 nucleotide long and was called KIN17(601). It contains an open reading frame coding for a 200 amino acid polypeptide. In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein. Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins.     

Analysis of the Escherichia coli K-12 cysB gene and its product using the method of gene fusion

The amino-terminal structure and the essential functional region of the cysB gene product of Escherichia coli K-12 were analyzed by the method of gene fusion. The translational start codon of the cysB gene was located by determining the amino-terminal sequence of a hybrid protein containing the first 31 amino acid residues of the CysB protein at the amino terminus of beta-galactosidase(LacZ protein). The fact that two other CysB'-'LacZ hybrid polypeptides expressed a normal CysB activity indicated that the functional region of the CysB protein was located within the first 215 amino acid residues of the total 324 amino acids deduced from the nucleotide sequence.     

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.     

Identification of low density lipoprotein receptor binding domains of human apolipoprotein B-100: a proposed consensus LDL receptor binding sequence of apoB-100

The human liver apoB-100 gene cloned in the lambda gt-11 expression vector expresses fusion proteins reacting with apoB antibodies. A fusion protein induced from a apoB-lambda gt-11 clone reacted with apoB-100 monoclonal antibodies known to block the binding of LDL to the LDL receptor. The fusion protein contains an amino acid sequence domain enriched in positively charged residues which is complementary to the negatively charged amino acids present in the consensus LDL receptor binding domain. This sequence of apoB-100 is proposed as a binding domain for the interaction with the LDL receptor. Comparison of derived amino acid sequences from the entire structure of apoB-100 molecule revealed several similar domains enriched in positively charged amino acids. A consensus sequence of the potential LDL binding domain was identified which contained positively charged amino acids at positions 1, 5 and 8 and a loop of 8-11 amino acids followed by two adjacent positively charged amino acids. These results are interpreted as indicating that there are several potential LDL receptor binding domains in apoB-100.     

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.