Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae.

The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Construction of an ori cassette for adapting shuttle vectors for use in Haemophilus influenzae.

An ori (origin of DNA replication) cassette, pORC, containing the P15a ori and the kanamycin-resistance-encoding gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which contains a promoterless chloramphenicol (Cm) acetyltransferase-encoding gene (cat) downstream from a multiple cloning site (MCS) [Brosius and Lupski, Methods Enzymol. 153 (1987) 54-68], to an E. coli-Haemophilus influenzae shuttle vector. The shuttle vector, pQL1, was shown to transform E. coli and H. influenzae efficiently. H. influenzae promoters were cloned into pQL1 by ligation of Sau3A-digested H. influenzae chromosomal fragments. Selection and semiquantitative analysis of promoter strength were performed on agar plates containing different concentrations of Cm. With the use of pQL1, H. influenzae gene regulation can now be studied in either H. influenzae or E. coli. In addition, elements of pORC can be used to convert other specialized E. coli vectors to E. coli-H. influenzae shuttle vectors.

Binding of human hemoglobin by Haemophilus influenzae.

Binding of biotinylated human hemoglobin to Haemophilus influenzae was detected when organisms were grown in heme-deplete, but not heme-replete, conditions. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled human hemoglobin or human hemoglobin complexed with human haptoglobin. Binding was only partially inhibited by rat hemoglobin, bovine hemoglobin, human globin, and bovine globin, and not at all by heme, human serum albumin, bovine serum albumin, human transferrin, or myoglobin. Hemoglobin binding was saturable at 16-20 ng of hemoglobin per 10(9) cfu. Binding of human hemoglobin was detected in serotypes a-f and serologically non-typable strains of H. influenzae, as well as Haemophilus haemolyticus but not Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus parahaemolyticus, or Escherichia coli.

The MA (p15) and p12 regions of the gag gene are sufficient for the pathogenicity of the murine AIDS virus.

Inoculation of the replication-defective retrovirus DEF27 (BM5d), packaged as an amphotropic virus pseudotype, into C57BL/6J mice leads to development of murine AIDS. Disease development showed a long incubation period (20 to 24 weeks), was associated with amplification of the BM5d provirus in splenocytes and lymph nodes, and was independent of the presence of exogenous or endogenous replication-competent helper viruses. However, both the onset of disease and amplification of the defective provirus were significantly enhanced by coinfection with the replication-competent B-cell-tropic ecotropic helper virus BM5e. The part of the BM5d viral genome that was essential for the pathogenicity was determined by making precisely engineered alterations in the reading frame of the gag and pol genes of BM5d proviral DNA and examining the ability of the altered amphotropic BM5d pseudotypes to induce the disease in C57BL/6J mice. The results show that expression of the MA (p15) and p12 regions of the gag gene is sufficient for pathogenicity of the BM5d retrovirus.

Dissociation between lymphoproliferative responses and virus replication in mice with different sensitivities to retrovirus-induced immunodeficiency.

Murine AIDS (MAIDS) is induced by a replication-defective virus (BM5d). In susceptible mice (C57BL/6J), inoculation with LP-BM5 murine leukemia virus, which consists of the BM5d virus and replication-competent B-tropic ecotropic (BM5e) and milk cell focus-inducing (BM5-MCF) helper viruses results in the polyclonal proliferation of T and B cells, immunodeficiency, and the expansion of B cells containing the BM5d provirus followed by the development of B-cell lymphomas. Several strains of mice that are resistant to LP-BM5-induced murine AIDS have been identified, and major histocompatibility complex genes as well as non-major histocompatibility complex genes were shown to play a role in this resistance. In the present study, we have examined and compared the replication of the BM5d and BM5e viruses after inoculation of LP-BM5 into sensitive (C57BL/6J) and resistant (C57BL/KSJ) mice. Using a specific polymerase chain reaction, we could detect the BM5d and BM5e proviruses as early as 1 week postinfection in the sensitive mice, and the levels of both viruses increased significantly with the progression of the disease. In contrast, in the resistant C57BL/KSJ mice, replication of BM5d and BM5e was restricted and no BM5d and only very low levels of the BM5e provirus could be detected either at early or late times postinoculation with the LP-BM5 virus mixture. Inoculation with LP-BM5 did not lead to the production of antibodies that could recognize the BM5d-encoded Pr60gag in either the sensitive or resistant mice; however, production of antibodies recognizing the env-related proteins of the helper virus was detected in the resistant but not in the sensitive mice at late times postinfection. Interestingly, inoculation with LP-BM5 increased polyclonal stimulation of spleen cells and decreased mitogen stimulation in both strains of mice. This stimulation of splenocytes persisted in the sensitive mice but decreased after a few weeks in the resistant mice. These results show an early block in BM5d and BM5e replication in the resistant C57BL/KSJ mice and indicate that resistance is a consequence of the inhibition of an onset of the BM5d virus infection and its expansion. However, initial responses to virus infection such as proliferation of spleen cells and response to mitogen are similar in both strains of mice and are therefore not necessarily related to the development of the disease.

In vivo generation of antigenic variants of murine retroviruses.

Inoculation of adult BALB/c-H-2k (BALB.K) mice with both Gross murine leukemia virus (GV) and a biological clone derived from this virus resulted in the recovery of variant viruses which differ from GV with respect to the expression of specific epitopes associated with the env gene product, gp70. The loss of these epitopes correlated with the failure of antiserum raised in BALB.K mice against GV to neutralize variant virus although this antiserum neutralized GV. In contrast, BALB/c-H-2b (BALB.B) mice, immunized with GV, produced antibodies which neutralized both GV and the variant virus, indicating that BALB.B mice respond to epitopes distinct from those recognized by BALB.K mice. These results suggest that the selection of variant viruses resulting from in vivo passage may be related to the immunoselective pressures exerted in mice which express particular alleles of certain major histocompatibility complex (MHC)-linked genes.

The human ribosomal RNA genes: structure and organization of the complete repeating unit.

The complete repeating unit of the human ribosomal RNA gene has been reconstructed by the cloning of approximately 27 kilobases (kb) of non-transcribed spacer. The structure of this tandemly repeated gene can now be studied in its entirety. We report the analysis of spacer DNA by molecular cloning and its organization in the genome by Southern transfer analysis. These studies reveal both length and sequence variation of the spacer. Sequence variations are distributed throughout the spacer while the length variations exist near the 5' end of the transcript and just beyond the 3' end. The human spacer shares extensive homology with primates but little with other mammals. Within the primates the degree of homology reflects the rapid evolutionary changes characteristic of the primate group.

Antigenic changes in gp70 associated with the adult variant of Gross murine leukemia virus, WB91.

Gross murine leukemia virus (GV) is not leukemogenic in adult mice whereas a variant of GV, WB91, is highly leukemogenic regardless of the age of the inoculated animal. FACS and SDS-PAGE analysis have demonstrated that these viruses differ at least with respect to the env-encoded gp70 molecule. FACS analysis of virus infected or virus transformed cells with a type specific monoclonal antibody (mAb #55) indicated a difference in determinants associated with gp70 expressed by the two viruses. Rat antisera raised against GV- or WB91-induced tumor cells demonstrated that there were no crossreactive determinants between the gp70 molecules expressed on these tumor cells as recognized by the rat antisera. This difference in the gp70 molecules encoded by WB91 and GV may account for the ability of the WB91 virus to induce leukemia in adult mice, possibly by affecting the immunogenicity of the virus.