The 45 kDa and 27 kDa yeast's protein kinases are not immunologically related.

Two yeast casein kinase type-1 species of 45 kDa and 27 kDa (CK1) were purified to apparent homogeneity and used for investigation of their immunological affinity. Antisera against the two kinases were isolated; the antibody against the 45 kDa kinase did not react with the 27 kDa enzyme. The 27 kDa casein kinase was recognized only by its own antibody. The obtained data strongly suggest that the low molecular mass CK-1 is not a proteolytic product of the 45 kDa kinase species.     

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up-regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.     

Yeast genomic clones encoding polypeptides immunologically related to an 18 kDa subunit of mitochondrial ATP synthase

A yeast genomic library in the bacteriophage expression vector lambda gt11 was screened with a polyclonal anti-holo-ATPase antiserum resulting in the isolation of 54 immunoreactive clones. Four of these phage clones express in bacteria a polypeptide antigenically related to an 18 kDa subunit (P18) of the yeast mitochondrial ATPase complex. Molecular analysis of the yeast DNA inserts in these phage clones revealed two classes of yeast DNA that share little homology at the nucleotide sequence level and therefore may represent distinct separate genes. The polypeptides potentially encoded by these yeast DNA segments do show scattered short blocks of strong amino acid sequence homology, which may underlie the observed immunochemical relatedness between the proteins expressed in bacteria.     

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare

Abstract Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M. intracellulare that is absent in M. avium . This antigen cross-reacts immunologically with a major 38 kDa antigen of M. tuberculosis , and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.     

Cloning and characterization of a gene encoding 22 kDa functional protein of bacteriophage MB78

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3'end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.     

P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity

A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property). Revision received: 11 August 1999 / Accepted: 8 September 1999     

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies     

A protein of Halobacterium halobium immunologically related to the v-myc gene product

A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.     

Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.     

A wheat gene encoding an aluminum-activated malate transporter

The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Al-dependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-activated malate transporter), that co-segregates with Al tolerance in F2 and F3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.     

Trichoplusia ni gloverin,an inducible immune gene encoding an antibacterial insect protein

By using differential display PCR, we obtained a cDNA clone encoding a gloverin homologue from the cabbage looper, Trichoplusia ni. The expression of the gene was induced by bacterial infections. The gene codes for a 174 amino acid residue protein, including a signal sequence and a prosegment. The deduced mature protein is 14 kDa and shows 58% and 49% identity to P2 from Helicoverpa armigera and to Hyalophora gloveri gloverin, respectively. The protein was detected in hemolymph and hemocytes from bacteria-immunized animals. We expressed gloverin using the baculovirus expression system. N-terminal amino acid sequence analysis showed that the purified protein contained a propart. This progloverin inhibited the growth of E. coli and the activity is comparable to that of H. gloveri mature gloverin. Processing of progloverin was possible in vitro, using human furin.     

A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation.

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.     

Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.     

Expression of the 18kDa protein of Mycobacterium leprae in Saccharomyces cerevisiae

Summary Mycobacterium leprae, the etiologic agent of leprosy, until now has not been grown in vitro, resulting in exceedingly obstacles for the production of purified antigens. It is therefore of interest to clone the relevant M.leprae antigens in other easy to handle microbial hosts. Here, we describe two different systems for expressing the 18kDa antigen of M.leprae in S. cerevisiae. Each system was shown to be effective in antigen expression, but the secretion system provided easier purification. Working with different host strains under different growth conditions, large quantities of biologically active proteins were obtained.     

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.