Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.     

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.     

A single point mutation resulting in an adversely reduced expression of DPM2 in the Lec15.1 cells

Mammalian dolichol-phosphate-mannose (DPM) synthase consists of three subunits, DPM1, DPM2, and DPM3. Lec15.1 Chinese hamster ovary cells are deficient in DPM synthase activity. The present paper reports that DPM1 cDNA from wild type and Lec15.1 CHO cells were found to be identical, and transfection with CHO DPM1 cDNA did not reverse the Lec15.1 phenotype. Neither did a chimeric cDNA containing the complete hamster DPM1 open reading frame fused to the Saccharomyces cerevisiae DPM1 C-terminal transmembrane domain. In contrast, Lec15.1 cells were found to have a single point mutation G29A within the coding region of the DPM2 gene, resulting in a glycine to glutamic acid change in amino acid residue 10 of the peptide. Moreover, mutant DPM2 cDNA expressed a drastically reduced amount of DPM2 protein and poorly corrects the Lec15.1 cell phenotype when compared with wild type CHO DPM2 cDNA (G(29) form).     

The divergent 5' ends of DPM2 mRNAs originate from the alternative splicing of two adjacent introns: characterization of the hamster DPM2 gene

Mammalian dolichol-phosphate-mannose (DPM) synthase has three subunits, DPM1, DPM2, and DPM3. In this report, an analysis of the gene and cDNAs of hamster DPM2 is presented. The CHO DPM2 gene has two special features. First, the initiation codon ATG is separated from the remainder of the coding region by intron sequences. Second, within these intron sequences the DPM2 gene contains an adjacent 3' splice site (acceptor) and a 5' splice site (donor), suggestive of a deleted exon between the first and second codons. In fact, these sites overlap by four nucleotides (nt) of AGGT. Splicing intermediates using both of these alternative splice sites were observed. This latter feature appears unique and is particularly unusual considering the relatively small size of the gene (2.7 kb) and of introns a (123 bp) and b (152 bp).     

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.     

The integrase family of tyrosine recombinases: evolution of a conserved active site domain.

The integrases are a diverse family of tyrosine recombinases which rearrange DNA duplexes by means of conservative site-specific recombination reactions. Members of this family, of which the well-studied lambda Int protein is the prototype, were previously found to share four strongly conserved residues, including an active site tyrosine directly involved in transesterification. However, few additional sequence similarities were found in the original group of 27 proteins. We have now identified a total of 81 members of the integrase family deposited in the databases. Alignment and comparisons of these sequences combined with an evolutionary analysis aided in identifying broader sequence similarities and clarifying the possible functions of these conserved residues. This analysis showed that members of the family aggregate into subfamilies which are consistent with their biological roles; these subfamilies have significant levels of sequence similarity beyond the four residues previously identified. It was also possible to map the location of conserved residues onto the available crystal structures; most of the conserved residues cluster in the predicted active site cleft. In addition, these results offer clues into an apparent discrepancy between the mechanisms of different subfamilies of integrases.     

Site-specific integration of the Haemophilus influenzae bacteriophage HP1. Identification of the points of recombinational strand exchange and the limits of the host attachment site.

Isotopic transfer experiments and boundary replacement studies were used to define the size and cleavage points of the Haemophilus influenzae attB site for phage HP1 integration. The points of strand cleavage and transfer were separated by 5' extensions with a spacing or overlap region most probably 7 residues long. The complete HP1 attB site is included within an 18-base pair (bp) sequence surrounding the cleavage sites. The sequence of HP1 attB is remarkably symmetric. Two 8-bp inverted repeats surround the central residue of the 7-bp overlap sequence; this central residue is the second residue of the anticodon sequence of the H. influenzae tRNA(leu)(UUR) gene which contains attB, and this symmetric segment encodes the anticodon stem and loop.     

Interaction of integration host factor from Escherichia coli with the integration region of the Haemophilus influenzae bacteriophage HP1.

The specific DNA-binding protein integration host factor (IHF) of Escherichia coli stimulates the site-specific recombination reaction between the attP site of bacteriophage HP1 and the attB site of its host, Haemophilus influenzae, in vitro and also appears to regulate the expression of HP1 integrase. IHF interacts specifically with DNA segments containing the att sites and the integrase regulatory region, as judged by IHF-dependent retardation of relevant DNA fragments during gel electrophoresis. The locations of the protein-binding sites were identified by DNase I protection experiments. Three sites in the HP1 attP region bound IHF, two binding sites were present in the vicinity of the attB region, and one region containing three partially overlapping sites was present in the HP1 integrase regulatory segment. The binding sites defined in these experiments all contained sequences which matched the consensus IHF binding sequences first identified in the lambda attP region. An activity which stimulated the HP1 site-specific integration reaction was found in extracts of H. influenzae, suggesting that an IHF-like protein is present in this organism.     

Site-specific integration of the Haemophilus influenzae bacteriophage HP1: location of the boundaries of the phage attachment site.

Plasmids containing DNA segments from the attachment region of phage HP1 were constructed and tested for the ability to replace the phage attachment site substrate in site-specific recombination reactions. The distance separating the boundaries of the functional site was 418 bp. Replacements within the 11-residue segment 5'-GGCGGTTATCG at the left boundary or within the 12-residue segment 5'-GGATTTTTTGAA at the right boundary abolished substrate activity. A segment of the 418-residue sequence preserves the integrity of an operon of three Haemophilus influenzae tRNA genes after HP1 insertion within the coding sequence.     

Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange

The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli , and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP -containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.