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A low G+C content genetic island in Mycobacterium avium subsp. paratuberculosis and M. avium subsp. silvaticum with homologous genes in Mycobacterium tuberculosis.

The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6.5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related 'genetic islands' and represents the first such element to be identified in mycobacteria.

Cell Wall↗

An in vitro ligation and transfection system for inserting DNA sequences into the latency-associated transcripts (LATs) gene of herpes simplex virus type 1.

This report describes a simple, rapid and highly efficient method for introducing specific DNA sequences into a defined locus of the herpes simplex virus type 1 (HSV-1) genome by restriction enzyme cleavage and ligation. The genome of the HSV-1 strain HFEM contains a 4.1 kb deletion in one copy of the RL region, deleting one copy of the latency-associated transcript (LAT) gene. It does not contain any site for restriction enzyme PacI. Two unique PacI restriction enzyme sites flanking an HSV-1 ICP6 promoter-LacZ reporter gene cassette were engineered into the LAT region to generate a recombinant virus HFEM/ICP6-LacZ which produced blue plaques in the presence of X-gal. This viral vector allowed the insertion of foreign genes directly into the HSV-1 genome by restriction enzyme digestion and ligation. The system was tested by digesting the HFEM/ICP6-LacZ DNA with PacI and with SwaI (an endogenous unique restriction enzyme site upstream of the LAT promoter locus and inserting by in vitro ligation a LAT promoter-LacZ gene cassette into the HFEM/ICP6-LacZ genome. The new recombinant virus HFEM/LAT-LacZ was detected as white plaques in the presence of X-gal, since beta-galactosidase expression, when driven by the LAT promoter, is not detectable during viral replication in tissue culture. The high yield (approximately 100%) of the recombinant virus obtainable from this in vitro ligation and transfection procedure coupled with a blue-white or reversible white-blue plaque detection scheme makes this a powerful method for constructing HSV-1 vectors around the LAT promoter locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Multiresistant Staphylococcus aureus: genetics and evolution of epidemic Australian strains.

Molecular and genetic analysis of multiresistant isolates of Staphylococcus aureus from widely separated hospitals in Australia has demonstrated that these are clearly related, and that the predominant strains possess up to three different plasmids, which fall into the following classes: (i) small 1.6 kb plasmids, such as pSK3, which are phenotypically cryptic, (ii) 4.5 kb chloramphenicol resistance plasmids, such as pSK2, and (iii) the pSK1 family of multiresistance plasmids, which range in size from 20 to 42 kb and variously encode resistance to antiseptics and disinfectants, trimethoprim (Tpr), penicillin (Pcr) and the aminoglycosides gentamicin, tobramycin and kanamycin (Gmr Tmr Kmr). Gmr Tmr Kmr is encoded on the pSK1 family plasmids by transposon Tn4001, which was also detected on the chromosomes of some clinical isolates. Tn4001 is composed of inverted repeats of the insertion sequence IS256; these repeats flank a Gmr Tmr Kmr sequence encoding for a 57,000 dalton bifunctional protein with aminoglycoside acetyltransferase [AAC(6')] and phosphotransferase [APH(2")] activities. A Tn4001-like structure, which is defective in transposition but encodes for a Gmr Tmr Kmr determinant homologous with that on Tn4001, occurs on conjugative plasmids from strains isolated in North America. Physical studies indicate that Pcr, via a beta-lactamase, and Tpr, via a trimethoprim-insensitive dihydrofolate reductase (DHFR), are also encoded on the pSK1 family by transposons; these transposons have been designated Tn4002 and Tn4003, respectively. Tn4003 is flanked by direct repeats of the insertion sequence IS257. The evolution of the pSK1 family of multiresistance plasmids is traced through the transposition and genetic rearrangement of resistance determinants. Transposition and genetic rearrangement have also contributed to the evolution of a multiresistant chromosome in Staph. aureus. In the majority of contemporary multiply resistant Staph. aureus strains the determinants for resistance to erythromycin (Emr), fusidic acid, methicillin (Mcr), minocycline, rifampicin, spectinomycin, streptomycin, sulphonamides, tetracycline (Tcr), cadmium (Cdr), and mercury (Hgr) are chromosomally encoded; these strains also possess chromosomally encoded Pcr, via a beta-lactamase. Evidence indicates that some of these determinants, Pcr, Cdr, Hgr, and Tcr, were plasmid encoded in isolates collected from Australian hospitals prior to 1970. Through transposition and site-specific integration, they have since been acquired by the chromosome in more recent Staph. aureus strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Australia↗

Molecular cloning and analysis of forked locus in Drosophila ananassae.

Drosophila ananassae, in spite of its unique genetic characters including high mutability and high frequency of male recombination has been little studied at the molecular level. Since the species is very similar to D. melanogaster, it is natural to expect that the high spontaneous mutation rate and male recombination may be caused by inserted mobile DNA elements, as in D. melanogaster. The present study was designed to determine whether or not most spontaneous mutations of the forked locus of D. ananassae are caused by insertion sequences as is found in D. melanogaster. I cloned the forked locus of D. ananassae, using forked DNA from D. melanogaster as a probe and investigated the molecular structure and transcription of the gene by Southern and Northern analyses. The results suggest that the restriction map and transcriptional patterns of the forked locus of D. ananassae are similar to those of D. melanogaster, while the spontaneous mutations available in D. ananassae are induced quite differently from those that have been described in D. melanogaster; in four (f;cd, f10-14, f49 and f86) out of eight forked alleles, neither insertions nor deletions were detected around the forked coding region. Forked transcripts are expressed in a pattern which is very similar to that of D. melanogaster and were all of normal size in these mutants. The other four mutants (f10-3, f9-10, f83i and f79b) had insertion sequences upstream of the forked coding region, while transcripts of the forked gene were of normal size. Hence, none of the eight mutations studied appear to affect the structure of the forked transcripts.

Animals↗

Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

Animals↗

Comparisons of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra bacilovirus mutants.

Few polyhedgra (FP) mutants of Autographa californica nuclear polyhedrosis virus (AcNPV) and the closely related strain Galleria mellonella (Gm)NPV have been reported which contain Trichoplusia ni host cell DNA sequences inserted into the viral genome between map units 35.0 and 37.7. New FP mutants are described with alterations of the HindIII-I restriction enzyme fragment (33.8 to 37.7 map units) of AcNPV, either deletions of viral DNA sequences or insertions of spodoptera frugiperda host cell DNA sequences. S. frugiperda DNA insertions from FP mutants were compared to T. ni host DNA insertions from FP mutants previously isolated (M.J. Fraser, G.E. Smith, and M.D. Summers (1983) J. Virol. 47, 287-300). Two host cell DNA sequences isolated from FP mutants, one of T. ni origin and one from S. frugiperda DNA, were transcribed in infected cells. Deletions of viral DNA sequences and insertions of host DNA sequences produce altered transcripts at the site of mutation as determined by both Northern and S1 nuclease analysis. Cell-free translation of cRNAs transcribed from wild-type viral DNA revealed an open reading frame coding for a 25-kDa protein at the site where host cell DNA insertions have been mapped. This was the same size as an infected-cell protein missing from most FP mutants examined.

DNA Transposable Elements↗

Identification of a contaminating Mycobacterium tuberculosis strain with a transposition of an IS6110 insertion element resulting in an altered spoligotype.

Molecular fingerprinting with the IS6110 insertion sequence is useful for tracking transmission of Mycobacterium tuberculosis within a population or confirming specimen contamination in the laboratory or through instrumentation. Secondary typing with other molecular methods yields additional information as to the relatedness of strains with similar IS6110 fingerprints. Isolated, relatively rare, random events within the M. tuberculosis genome alter molecular fingerprinting patterns with any of the methods; therefore, strains which are different by two or more typing methods are usually not considered to be closely related. In this report, we describe two strains of M. tuberculosis, obtained from the same bronchoscope 2 days apart, that demonstrated unique molecular fingerprinting patterns by two different typing methods. They were closely linked through the bronchoscope by a traditional epidemiologic investigation. Genetic analysis of the two strains revealed that a single event, the transposition of an IS6110 insertion sequence in one of the strains, accounted for both the differences in the IS6110 pattern and the apparent deletion of a spacer in the spoligotype. This finding shows that a single event can change the molecular fingerprint of a strain in two different molecular typing systems, and thus, molecular typing cannot be the only means used to track transmission of this organism through a population. Traditional epidemiologic techniques are a necessary complement to molecular fingerprinting so that radical changes within the fingerprint pattern can be identified.

Bacterial Typing Techniques↗

IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.

Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.

DNA Nucleotidyltransferases↗

Genetic analysis of capsular status of meningococcal carrier isolates.

The meningococcal capsule is the primary virulence factor with systemic isolates requiring full expression of the capsule but with capability to down-regulate the capsule in order to invade. The meningococcal capsular operon is composed of a number of genes that are involved in capsular synthesis and transport. Differences in capsular synthesis genes may allow discrimination between meningococcal serogroups whereas absence of genes for either synthesis or transport imply that the meningococcus is unencapsulated. Although mechanisms such as slipped-strand mispairing and acquisition of insertion sequences have been demonstrated to be involved in regulation of capsular expression, few studies have addressed the mechanisms of capsular expression in carrier isolates. Following a community-based intervention programme for an outbreak of meningococcal disease, we collected meningococcal carrier isolates from the intervention area and control areas. We undertook genetic analysis of the capsular operon and the mechanisms of capsular regulation, together with an investigation of the potential of capsular genes to identify the genogroup of non-serogroupable isolates. Use of the siaD gene allowed the discrimination of 30/89 (34%) non-serogroupable isolates into B, C, W135 and Y with a siaA gene PCR permitting the characterization of a further 6 isolates whose capsules contained sialic acid. Slipped-strand mispairing was evident in only 4 of 13 genogroupable B isolates and the insertion sequence IS1301 was found in 2 of 36 siaA-positive isolates. Of 51 non-genogroupable isolates 25 (49%) were shown to be ctrA negative. There was a higher percentage of ctrA-positive isolates (P<0.001) amongst meningococcal strains obtained from those sampled in non-intervention schools than those sampled at intervention schools. The ctrA-negative isolates warrant further investigation of their genotypic organization since such avirulent strains may be important in conferring natural protection against invasive disease. We found that after mass antibiotic prophylaxis, recolonization occurs preferentially with non-pathogenic meningococcal strains. This as implications for assessment of the benefits of mass antibiotic and vaccination programmes for outbreak control. Previously expressed concerns of increased risk due to removal of protective ora may have been overstated.

Bacterial Capsules↗

Association of polymorphisms for prolactin and prolactin receptor genes with broody traits in chickens.

Prolactin (PRL) is generally accepted as crucial to the onset and maintenance of broodiness in avian species. The prolactin receptor (PRLR) plays an important role in the PRL signal transduction cascade. Two candidate genes, PRL and PRLR, were screened for polymorphisms in the chicken, and their genetic effects on broodiness were evaluated. Pedigreed hens (n = 155) of the Blue-shell chicken, a Chinese local breed, were observed for phenotypic broody traits including nesting days, broody days, repeats of broody cycles, and duration of broodiness. For polymorphism analysis, White Leghorns, Hy-Line brown egg layers, Avian broilers, and some other Chinese local breeds were included. Fifteen sets of primers were used to amplify the nucleotide sequences of the promotor of PRL and exons of PRLR. The PCR products were screened for polymorphisms using single-stranded conformational polymorphism protocol. Sequencing revealed a 24-bp insertion occurring in the promotor, -377 approximately -354, of PRL (GenBank accession no. AB011434). A single nucleotide polymorphism (SNP), A9026G (GenBank accession no. AY237377), in exon 3 of PRLR was also detected, which led to a nucleotide transition in the 5'-untranslated region (5'-UTR) of PRLR cDNA. Two SNP, T14771C and G14820A (GenBank accession no. AY237376), were detected in exon 6 of the PRLR. The T14771C transition led to an amino acid variation, Leu340Ser, in PRLR, whereas the G14820A transition was a synonymous mutation. An association analysis showed that the genetic polymorphisms at PRLR3 and PRLR6 were not related to broodiness (P > 0.05), whereas the individuals without the insertion sequence at PRLpro2 were associated with broody traits (P < 0.05) and the incidence (>30%) of typical broody of genotypes +/- and -/- was higher (P < 0.01) than that of +/+. In addition, all White Leghorns were +/+ for PRLpro2, whereas local breeds with very strong broodiness were nearly all -/-. Homozygous insertion of the 24-bp sequence in the PRL promoter may decrease the expression of PRL, leading to nonbroodiness. The results suggested that PRLpro2 could be a genetic marker in breeding against broodiness in chickens.

Animals↗

Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.

A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.

Bordetella↗

Regulated expression of glutamyl-tRNA synthetase is directed by a mobile genetic element in the cyanobacterium Tolypothrix sp. PCC 7601.

The genome of Tolypothrix sp. PCC 7601 carries two copies of a novel insertion sequence, ISTosp1. One of the two copies is located upstream of the gene encoding glutamyl-tRNA synthetase, an enzyme playing a key role in protein and pigment synthesis. The tnpA gene of the IS element and gltX were co-transcribed and their expression was transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source were available. The second copy is also transcribed and shows a similar regulatory pattern. Structural elements of the promoter (-10 and -35 sequences) directing the expression of the tnpA-gltX operon have been localized within the IS. Regulatory sequences involving the NtcA transcription factor in the control of tnpA-gltX expression were found both within and in sequences upstream of the insertion element. The expression of gltX in a closely related cyanobacterium, Nostoc sp. PCC 7120, which lacks the insertion upstream of gltX, decreased upon ammonium retrieval, a regulatory pattern that markedly differs from that observed in Tolypothrix sp. PCC 7601. ISTosp1 constitutes a good example of how cells can make use of a transposable element to evolve an original regulatory mechanism.

Bacterial Proteins↗

hsk1+, a Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal replication.

Degenerate oligonucleotide-directed polymerase chain reaction was conducted to clone a possible Schizosaccharomyces pombe homologue [hsk1 for a putative homologue of CDC7 (seven) kinase 1] of Saccharomyces cerevisiae Cdc7 kinase. The cloned cDNA for hsk1+ contains an open reading frame consisting of 507 amino acids with predicted mol. wt of 58,370 that possesses overall amino acid identity of 46% (65% including similar residues) to CDC7. In addition to conserved domains for serine-threonine kinases, the predicted primary structure of Hsk1 contains three 'kinase insert' sequences characteristic to Cdc7 at the positions identical to those of Cdc7. Whereas the length and sequences of the kinase inserts are diverged between the two yeast species, 58% identity (76% including similar residues) is detected within the kinase conserved domains. The hsk1+ gene, which is present as a single copy on the S.pombe chromosome, contains two introns within the coding frame. Disruption of the hsk1+ gene by insertion of the ura4+ gene is lethal to growth. Analysis of the DNA content of germinating spores that contain hsk1 null alleles indicates that DNA replication is inhibited in the mutant. The morphology of these mutant spores after germination indicates abnormal nuclear division in some population of germinating spores, suggesting either that Hsk1 may be required for inhibition of mitosis until completion of S phase or that it may also be involved in proper execution of mitosis. Our results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.

Amino Acid Sequence↗

Preparation of triple-block DNA polymers using recombinant DNA techniques.

The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described. The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase. The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E. coli. Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28. Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site. The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts. Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally.

Animals↗

Characteristics of IS401, a new member of the IS3 family implicated in plasmid rearrangements in Pseudomonas cepacia.

We have determined the nucleotide sequence of IS401, an insertion sequence implicated in rearrangements of a 170-kb cryptic plasmid from Pseudomonas cepacia. Our analysis focused on a 4066-bp plasmid fragment containing adjacent copies of IS401 and of IS408, an element reported previously to activate gene expression in P. cepacia. One objective was to determine if an apparent increase in the copy number of IS401 in strains carrying adjacent plasmid copies of these two elements might be due to readthrough transcription of an IS401 transposase gene from an outwardly directed promoter within IS408. This possibility was ruled out by nucleotide sequence analysis of the 4066-bp plasmid fragment, which indicated that the major open reading frames of IS401 were oriented in the direction of IS408. IS401 was 1316 bp in length and had 26-bp terminal inverted repeats flanked by 3-bp direct duplications of adjacent DNA. It was closely related to the IS3 family elements IS51 from P. savastanoi and IS3411 from Escherichia coli. Pertinent features of IS408 are also discussed.

Amino Acid Sequence↗

Codon usage and G + C content in Bradyrhizobium japonicum genes are not uniform.

To date, the sequences of 45 Bradyrhizobium japonicum genes are known. This provides sufficient information to determine their codon usage and G + C content. Surprisingly, B. japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G + C content than genes not belonging to these two groups. Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes. The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj alpha 9 sequence which is an insertion sequence (IS)-like element. The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors. These were now corrected. The deduced gene product of ORF355 in RSRj alpha 9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes. The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements.

Amino Acid Sequence↗

Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37.

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.

Amino Acid Sequence↗