Immunological detection of sheep experimentally infected with strains of Mycobacterium avium subspecies containing insertion sequence IS901/IS902 and a 40 kDa protein

A monoclonal antibody raised against a 40 kDa protein present in certain M. avium strains (IS901/IS902 positive) was used for developing a blocking ELISA. Sera from experimentally infected sheep were evaluated by indirect ELISA, AGID and blocking ELISA. The blocking assay proved to be highly specific for differentiation of sheep infected with different subspecies of M. avium.     

Stability of insertion sequence IS1245, a marker for differentiation of Mycobacterium avium strains

Recently a novel insertion element, IS1245, has been described and suggested for use as a probe in restriction fragment length polymorphism studies of Mycobacterium avium strains. An important issue in this context is the stability of the insertion element. We analyzed single colonies of M. avium cultures and found frequent small one- to two-band changes. However, following repeated in vitro passages over 1 year, similar one- to two-band changes were observed in the IS1245 patterns of only six M. avium strains investigated.     

Structure of the gene encoding the alpha subunit of the human interleukin 3 receptor

Interleukin 3 is a cytokine that stimulates proliferation and differentiation of hematopoietic progenitor cells. Its receptor consists of two subunits, an interleukin 3-specific alpha subunit and a beta subunit shared by garanulocyte-macrophage colony stimulating factor and interleukin 5 receptors. In this paper, we determined the genomic structure of the alpha subunit of the human interleukin 3 receptor, which spans approximately 40 kb and has 12 exons. We found that the genomic structures of the alpha subunits of the human interleukin 3 and granulocyte-macrophage colony stimulating factor receptors are very similar. They possess a unique additional intron in the 'C domain', which is absent in the alpha subunit of the interleukin 5 receptor. These results suggest a shared evolutionary pathway of these two genes.     

Sequence-based differentiation of strains in the Mycobacterium avium complex

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Characterization of the gene encoding the catalytic subunit of the DNA polymerase alpha from Toxoplasma gondii

The gene encoding the catalytic subunit of the Toxoplasma gondii DNA polymerase alpha enzyme has been isolated. The coding region is 6487 bp in length, containing three introns, and specifies a protein of 1690 aa. The seven conserved regions which characterize the polalpha polypeptide, as well as four of the five polalpha-specific aa domains, were found in the T. gondii gene. The absence of one of these domains, as well as the presence of a unique cysteine cluster between domains IV and B in the T. gondii polalpha, may result in a slight difference in the secondary or even tertiary structure compared with the human homologue and thus may be suitable for designing anti-Toxoplasma drugs. A number of amino acid differences within the seven conserved regions between the human and T. gondii polalpha, as well as variations in the spacings of these regions, were also observed.     

Identification of adjacent genes encoding the major catalase and a bacterioferritin from the plant-beneficial bacterium Pseudomonas putida

The glycosides in mono-, di- and trihydroxylated terpene and norisoprenoid alcohols and also those in the related shikimate pathway have been isolated on C18 reversed-phase cartridges and then fractionated into classes of different polarity at increasing percentages of methanol. The benzyl alcohol glycosides are the most polar, while those of terpene monohydroxylated alcohols and geranic acid are the least polar. The terpene diols, linalool furanoid and pyranoid oxides and also norisoprenoid precursors show intermediate polarity and separate into well defined fractions according to their polarity.     

Complete sequence of a hair-like intermediate filament type II keratin gene

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO2 fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.     

Mycobacterium avium complex and Mycobacterium tuberculosis in patients infected with the human immunodeficiency virus

Primary care physicians play an important role in identifying and treating bacterial infections in adults infected with the human immunodeficiency virus (HIV). Mycobacterium avium complex and Mycobacterium tuberculosis are pathogens that can cause systemic or local infection in these patients. We review the epidemiology, pathogenesis, clinical presentation, and principles of treatment for these two mycobacterial pathogens. Because M tuberculosis disease is preventable and curable and yet communicable, physicians should maintain a high degree of suspicion for tuberculosis in HIV-infected adults. In comparison, the goal of treating M avium complex in patients with advanced HIV disease is to reduce constitutional symptoms and improve survival.     

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.