ClaI Fragment Research Articles

Bacteriophage-Encoding Cytolethal Distending Toxin Type V Gene Induced from Nonclinical Escherichia coli Isolates

Cytolethal distending toxin (Cdt) is produced by a variety of pathogenic bacteria, including pathogenic serotypes of Shiga toxin-producing Escherichia coli (STEC). The Cdt family comprises five variants (Cdt-I to Cdt-V) encoded by three genes located within the chromosome or plasmids or, in the case of Cdt-I, within bacteriophages. In this study, we evaluated the occurrence of the cdt gene in a collection of 140 environmental STEC isolates. cdt was detected in 12.1% of strains, of which five strains carried inducible bacteriophages containing the Cdt-V variant. Two Cdt-V phages of the Siphoviridae morphology lysogenized Shigella sonnei, generating two lysogens: a single Cdt phage lysogen and a double lysogen, containing a Cdt phage and an Stx phage, both from the wild-type strain. The rates of induction of Cdt phages were evaluated by quantitative PCR, and spontaneous induction of Cdt-V phage was observed, whereas induction of Stx phage in the double lysogen was mitomycin C dependent. The Cdt distending effect was observed in HeLa cells inoculated with the supernatant of the Cdt-V phage lysogen. A ClaI fragment containing the cdt-V gene of one phage was cloned, and sequencing confirmed the presence of Cdt-V, as well as a fragment downstream from the cdt homolog to gpA, encoding a replication protein of bacteriophage P2. Evaluation of Cdt-V phages in nonclinical water samples showed densities of 10(2) to 10(9) gene copies in 100 ml, suggesting the high prevalence of Cdt phages in nonclinical environments.

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

Nonsense Suppression in Yeast Cells Overproducing Sup35 (eRF3) Is Caused by Its Non-heritable Amyloids

The [PSI+] prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils. Prion state of the Rnq1 protein, [PIN+], is required for the [PSI+] de novo generation but not propagation. Yeast [psi-] [PIN+] cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable [PSI+]. Here, we show that in such cells, most of Sup35 represents amyloid polymers, although the remaining Sup35 monomer is sufficient for normal translation termination. The presence of these polymers strictly depends on [PIN+], suggesting that their maintenance relies on efficient generation de novo rather than inheritance. Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization. About 10% of cells overproducing Sup35 form colonies on medium selective for suppression, which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants, allowing selection of cells deficient for soluble Sup35. A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced. The described polymers of these proteins differ from the [PSI+] polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.

Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum (ER)-associated Degradation and Sensitivity to ER Stress

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.

A non-antibiotic marker for amplification of plant transformation vectors in E. coli

Concern over perceived risks from the presence of antibiotic resistance genes in transgenic plants is leading to selection strategies which do not use antibiotic resistance genes as selectable markers. The concern regarding the presence of antibiotic resistance genes has focused particularly on the bla gene for ampicillin resistance, which is used as a bacterial marker when pUC-based plasmids are amplified prior to microprojectile bombardment. The rtl operon, which permits the use of ribitol as the sole carbon source, is present in Escherichia coli strain C but absent in all the K-12 laboratory strains commonly used for molecular biology. A ClaI fragment containing the kinase, dehydrogenase and transporter components of the rtl operon was isolated from strain C and subsequently used to replace bla from two cloning vectors, pBluescript and pMECA, to create pBluescript-R and pMECA-R. E. coli K-12 strain DH10B transformed with either plasmid acquired the ability to grow on ribitol as the sole carbon source, as long as pMECA-R was present within the bacterial cell. Plasmid yields were evaluated and found to be comparable to those obtained from bacteria growing on LB medium supplemented with ampicillin. Hence, the use of genes from the rtl operon might be an acceptable alternative to the use of bla.

Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) fromClostridium bifermentansDPH-1

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.

The Structural Protein E of the Archaeal Virus φCh1: Evidence for Processing in Natrialba magadii during Virus Maturation

φCh1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii. The virus morphology resembles other members of Myoviridae infecting Halobacterium species. The gene of the major capsid protein E of virus φCh1 was cloned and the DNA sequence was determined. Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5′-end of the φCh1 genome. The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame. We present evidence that the gene E product is proteolytically cleaved between Lys16 and Asn17 to yield the 305 residue polypeptides found in the mature viral capsid. Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies. Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of φH, infecting Halobacterium salinarum. RT-PCR analysis as well as Western blot studies revealed gene E as a late gene. Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N. magadii L11.

Delivery of a Retroviral Vector Expressing Human β-Glucuronidase to the Liver and Spleen Decreases Lysosomal Storage in Mucopolysaccharidosis VII Mice

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

In Vitro Studies on tRNA Annealing and Reverse Transcription with Mutant HIV-1 RNA Templates

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semistable stem-loop structure, the U5-PBS hairpin, which occludes part of the tRNA primer binding site (PBS). In previous studies, we demonstrated that mutations that alter the stability of the U5-PBS hairpin inhibit virus replication. A reverse transcription defect was measured in assays with the virion-extracted RNA-tRNA complexes. We now extend these studies with in vitro synthesized wild-type and mutant RNA templates that were tested in primer annealing and reverse transcription assays. The effect of annealing temperature and the presence of the viral nucleocapsid protein on reverse transcription was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to stable template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA primed reactions. The inhibitory effect of template structure on tRNA-primed reverse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex differs from the virion-extracted viral RNA-tRNA complex.

The Tyrosines in the Bidentate Motif of the env-sea Oncoprotein Are Essential for Cell Transformation and Are Binding Sites for Grb2 and the Tyrosine Phosphatase SHP-2

The transforming gene product of the S13 avian erythroblastosis virus, the env-sea protein, is a member of the hepatocyte growth factor receptor family of tyrosine kinases comprising Met, Ron, and Sea. Like all three members of this family, the env-sea protein has a so-called bidentate motif (Y557INMAVTY564VNL) composed of two tandemly arranged tyrosines in the carboxyl terminus. To investigate whether the tyrosine residues in this motif are essential for the env-sea-mediated transformation, we generated tyrosine to phenylalanine mutations. Substitutions of both tyrosine residues resulted in complete loss of the transforming activity. In contrast, single mutations at either tyrosine did not inhibit transformation of Rat1 cells, and mutation of tyrosine 564 actually increased transformation of Rat 1 cells. To define signaling pathways activated by the env-sea protein, we looked for protein-protein interactions mediated by these tyrosine residues. We show that the bidentate motif is responsible for interaction with the adapter protein Grb2, phosphatidylinositol 3-kinase, and the tyrosine phosphatase SHP-2. Furthermore, we show that microinjected Src homology 2 domains from either Grb2 or SHP-2 blocked the transforming activity of the env-sea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation.

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge.

The number of kringle IV repeats 3–10 is invariable in the human apo(a) gene

The human apolipoprotein(a) ( apo(a)) gene is a member of a family of related genes including plasminogen, apo(a)rg-B and apo(a)rg-C, which are clustered on chromosome 6q 2,7. Apo(a) contains ten different types of plasminogen-like kringle IV repeats (K-IV 1–10) one of which (K-IV 2) varies in number resulting in a remarkable size polymorphism of the protein. Sequence analysis of human apo(a) alleles and indirect evidence have suggested that K-IV 1 and K-IV 3–10 are each present once in individual alleles and that the 3′ apo(a) region encompassing kringles IV 3–10, kringle V and the protease domain is invariable. To directly test this, we have constructed a restriction map of the apo(a) gene region from genomic DNA and from a yeast artificial chromosome (YAC) (K-IV 13) which contains the entire apo(a) gene. The presence of a 63 kb ClaI fragment encompassing kringles IV 3–10, kringle V and the protease domain and a 46 kb SwaI fragment, spanning kringles IV 5–10, kringle V and the protease domain was demonstrated by PFGE/Southern blotting in 30 unrelated subjects, who represented a range of apo(a) size alleles containing from 11 to 49 kringles. Our analysis demonstrates that the number of kringles IV 3–10 is invariable in the human apo(a) gene, suggesting that the 3′ domain of Apo(a) is functionally important.

Two protein-tyrosine phosphatases inactivate the osmotic stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1.

Protein phosphatases inactivate mitogen-activated protein kinase (MAPK) signaling pathways by dephosphorylating components of the MAPK cascade. Two genes encoding protein-tyrosine phosphatases, PTP2, and a new phosphatase, PTP3, have been isolated in a genetic selection for negative regulators of an osmotic stress response pathway called HOG, for high osmolarity glycerol, in budding yeast. PTP2 and PTP3 were isolated as multicopy suppressors of a severe growth defect due to hyperactivation of the HOG pathway. Phosphatase activity is required for suppression since mutation of the catalytic Cys residue in Ptp2 and Ptp3, destroys suppressor function and biochemical activity. The substrate of these phosphatases is likely to be the MAPK, Hog1. Catalytically inactive Ptp2 and Ptp3 coprecipitate with Hog1 from yeast extracts. In addition, strains lacking PTP2 and PTP3 do not dephosphorylate Hog1-phosphotyrosine as well as wild type. The latter suggests that PTP2 and PTP3 play a role in adaptation. Consistent with this role, osmotic stress induces expression of PTP2 and PTP3 transcripts in a Hog1-dependent manner. Thus Ptp2 and Ptp3 likely act in a negative feedback loop to inactivate Hog1.

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.

Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene.

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species.

The molecular cloning of genes specifying some enzymes of the 3,5-xylenol degradative pathway

Enzymes for the degradation of 3,5-xylenol ofPseudomonas putida NCIB9869 are encoded on a transmissible plasmid, pRA500. Genes specifying the inducible synthesis of some enzymes encoded on pRA500, namely, citraconase (G), citramalate coenzyme-A-transferase (H), citramalyl-coenzyme-A lyase (I), maleyl-pyruvate isomerase (J) and fumarylpyruvate hydrolase (K) have been cloned on a 7.9-kbHindIII fragment into the vector pKT231 to give pRA507. Biochemical and restriction analysis of pRA507 and some of its deleted derivatives has enabled preliminary locations to be assigned to the genes encoding these enzymes. The structural genes for enzymes H and I, together with a regulatory gene controlling their expression, are located on a 1.6-kbHindIII/XhoI fragment of pRA507 and the genes for enzymes G, J and K are located within a 4.5-kbXhoI/ClaI fragment of pRA507, which lies immediately adjacent to the former fragment. Biochemical analysis of the strain carrying pRA507, of its plasmid-free derivative and of other plasmid-free derivatives of the wild-typeP. putida has provided indirect evidence for the presence of two isofunctional enzymes at three different steps of the degradative pathway. One set of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase and maleylpyruvate hydrolase is encoded on pRA500 and these enzymes are inducible only. A second set is encoded on the chromosome and these enzymes are constitutive and not further inducible. The chromosomally located genes appeared to be silent until 3,5-xylenol-negative strains were incubated for prolonged periods with compounds such asm-hydroxybenzoate orp-cresol.

The Amino-terminal One-third of αIIb Defines the Ligand Recognition Specificity of Integrin αIIbβ3

The integrin alpha subunits play a major role in the regulation of ligand binding specificity. To gain further insight into the regions of the alpha subunits that regulate ligand specificity, we have utilized alpha v / alpha IIb chimeras to identify regions of alpha IIb that when substituted for the homologous regions of alpha v switched the ligand binding phenotype of alpha v beta 3 to that of alpha IIb beta 3. We report that the ligand recognition specificity of beta 3 integrins is regulated by the amino-terminal one-third of the alpha subunit. Substitution of the amino-terminal portion of alpha v with the corresponding 334 residues of alpha IIb reconstituted reactivity with both alpha IIb beta 3-specific activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies in addition to small highly specific activation-independent ligands. In contrast, substitution of the amino-terminal portion alone or the divalent cation repeats alone were not sufficient to change ligand binding specificity. These data in combination with previous studies demonstrate that integrin ligand recognition requires cooperation between elements in both the alpha and beta subunits and indicate that the ligand binding pocket is a structure assembled from elements of both the alpha and beta subunits.

Chromosomal organisation of a repeated gene cluster expressed in mammalian stages of Leishmania

The genomic organisation of a large Leishmania gene cluster, expressed predominantly in intracellular, infective parasite stages, has been determined. Using cosmid cloning, parasite DNA fingerprinting, partial digestion and mapping with ‘end-specific’ probes, the LmcDNA2 gene array has been localised to a 55-kb ClaI fragment within the L. major genome. Six tandemly linked gene copies are transcribed to produce an abundant 6-kb transcript; the seventh and last copy of the cluster is truncated at its 3′ end. It is likely that these genes encode one or more proteins specific to infective stages of the parasite life cycle.

Molecular analysis of an A-protein secretion mutantof Aeromonas salmonicida reveals a surface layer-specific protein secretion pathway

The Aeromonas salmonicida Tn5 mutant, A449-TM1, is unable to secrete thesurface layer protein (A-protein) through the outer membrane. Immunogold labeling of thin sections of A449-TM1, with polyclonal antisera against the A-protein, showed the accumulation of large quantities of A-protein in an enlarged periplasm. The majority of the labeled A-protein could be seen at the poles of the cells. The ability of A449-TM1 to secrete other extracellular proteins such as hemolysin and protease was not impaired by the Tn5 insertion, which indicates that the mutation in A449-TM1 interferes with a secretion pathway specifically for the translocation of the A-protein through the outer membrane. The mutant, A449-TM1, was shown to be avirulent for fish. A cosmid clone from a gene library of A449-TM1, which contains the Tn5 insertion from the chromosome, was used to identify a 1.4 kb SaII/ ClaI fragment from immediately adjacent to the Tn5 insertion. This fragment was used to identify and clone a 4 kb HindIII fragment from a chromosomal DNA digest from the wild-type strain, A449. DNA sequence analysis of this clone identified an open reading frame (ORF) of 1656 bp. The deduced product of this ORF showed sequence similarity to a family of ATP-binding secretion proteins, but appeared to be phylogenetically distinct from these proteins, consistent with its participation in a secretory pathway specific for surface layer protein.