Expression of gonococcal protein II in Escherichia coli by translational fusion

A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.     

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.     

A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.     

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.     

Identification and characterization of the products from the traJ and traY genes of plasmid R100.

The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with beta-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.     

Regularities of the nucleotide sequence at the 5'-end of the codon in Escherichia coli genes

The frequencies of occurrence of nucleotides at the 5' side of codons have been determined in highly and weakly expressed genes from E. coli. Significant constraints on the nucleotide 5' to some codons were found in highly expressed genes. Certain rules of synonymous codon usage depending on the amino acid 3' of the codon were established. E. g., codon possessing quanosine in the third position (NNG) are preferred over NNA if the next amino acid is lysine (P less than 10(-5)). On the other hand, rules of synonymous codon usage in relation to 5' flanking nucleotide were found. For example, when coding for aspartic acid, GAC codon is preferred over GAU (P less than 0.001) if uridine is 5' to codon and on the contrary GAU is favoured (P less than 0.0001) if quanosine is at the 5' side of aspartic acid codon. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5

The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da.     

Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli

Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.     

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.     

Libraries of green fluorescent protein fusions generated by transposition in vitro

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.     

Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis.

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.