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PubMed1991

Three-dimensional structures of acidic and basic fibroblast growth factors.

Members of the fibroblast growth factor (FGF) family of proteins stimulate the proliferation and differentiation of a variety of cell types through receptor-mediated pathways. The three-dimensional structures of two members of this family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. The locations of sequences implicated in receptor and heparin binding by FGF are presented. These sites include beta-sheet strand 10, which is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family, and which shows sequence conservation among the FGF family and interleukin-1 beta.

Amino Acid Sequence↗

PubMed1998

Microbiology of a nitrite-oxidizing bioreactor.

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G + C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer Nitrospira moscoviensis. The NOSBR sludge was overwhelmingly dominated by bacteria closely related to N. moscoviensis (89%). Two clone sequences were similar to those of the genus Nitrobacter. Near-complete insert sequences of eight RC and one GC N. moscoviensis clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the Nitrospira phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.

Bacteria↗

PubMed1988

Isolation and comparison of Escherichia coli strains from canine and human patients with urinary tract infections.

We analyzed Escherichia coli strains isolated from dogs with urinary tract infections (UTIs) in an attempt to determine if any of these strains were similar to E. coli isolated from humans with UTIs. Using genotypic and phenotypic traits, we identified four canine and six human E. coli UTI isolates that all appeared to be closely related or identical. All isolates shared similar DNA sequences for pyelonephritis-associated pili (pap), alpha-hemolysin (hly), and insertion sequence 5 (IS5), on the basis of Southern blot analysis. Similar outer membrane protein, pilin, and plasmid profiles were obtained for each of the isolates, although minor heterogeneity was observed. All of these isolates expressed a neuraminidase-sensitive binding phenotype in contrast to the majority of human isolates, which are known to express an adhesin that recognizes terminal digalactoside residues. Taken together, these results suggest that similar E. coli uropathogens may be capable of infecting both dogs and humans. To determine if the intestinal tracts of dogs were a reservoir for uropathogenic E. coli, eight paired rectal and urine pap+ E. coli strains were cultured from dogs with UTIs. By using the same genotypic and phenotypic criteria described above as a basis for strain identity, seven of eight urine-rectal pairs showed intrapair identity. However, each urine-rectal pair displayed a unique overall profile and could be distinguished from the other pairs. We conclude that the uropathogen colonizing the bladders of dog can also be the predominant strain colonizing the intestinal tracts.

Animals↗

PubMed1999

Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain.

Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a approximately 7.9-kb DraI fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS6110 contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110 insertion in M. tuberculosis H37Ra near oriC. Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.

Animals↗

PubMed2000

Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region.

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain chi7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with chi7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain chi7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain chi7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

Adhesins, Escherichia coli↗

PubMed1999

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome.

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.

Adenosine Triphosphatases↗

PubMed2000

Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes.

Amino Acid Sequence↗

PubMed1989

Isolation and characterization of cDNA clones encoding embryonic and adult isoforms of rat cardiac troponin T.

We have previously reported that troponin T (TnT), a regulatory protein in muscle contraction, undergoes a switch from the larger, acidic embryonic form to the smaller, basic adult form during rat heart development (Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309-7315). To investigate the significance and the molecular mechanism of this isoform switching, cDNA clones encoding rat cardiac TnT were obtained by screening a cDNA library constructed from young rat cardiac poly(A)+ RNAs using the expression vector lambda gt11. Clone RCT10 proved to be a full length clone containing 50 base pairs (bp) of 5'-untranslated sequence, 870 bp of coding sequence, and 196 bp plus poly(A) tail at the 3'-untranslated region. In the other cDNA clone (RCT11), the entire 3'-untranslated sequence and most of the coding sequence were identical with that of RCT10, but an additional 30-bp insert was present in the coding region from residues 18 to 27. This insertion sequence appeared to code for a fragment (EDWSEEEEDE) highly enriched in acidic amino acid residues. Thus, RCT11 might represent a clone encoding the embryonic isoform of rat cardiac TnT. S1 nuclease RNA mapping analysis using end-labeled RCT11 cDNA probes confirmed that this region of the sequence is different in embryonic and adult isoform mRNAs. These results suggest that both embryonic and adult isoforms of rat cardiac TnT are generated from the same primary transcript by developmentally regulated alternative splicing. The amino acid sequence deduced from RCT10 cDNA exhibits 87%, 85%, and 72% homology with bovine, rabbit, and chicken cardiac TnTs, respectively, but less homology (57-59%) with the known skeletal TnTs from human, rat, rabbit, and chicken. Moreover, both the 5'- and the 3'-untranslated sequences of rat cardiac TnT mRNA are completely different from those reported for rat skeletal TnT mRNA, suggesting that rat cardiac TnT is coded from a gene distinct from the rat skeletal TnT gene.

Aging↗

PubMed1993

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Base Sequence↗

PubMed1995

Positions of multiple insertions in SSU rDNA of lichen-forming fungi.

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.

Base Sequence↗

PubMed1995

Three region-specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24.

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70-94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.

Amyotrophic Lateral Sclerosis↗

PubMed1996

IS1394 from Pseudomonas alcaligenes N.C.I.B. 9867: identification and characterization of a member of the IS30 family of insertion elements.

A new insertion sequence designated IS1394 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) by entrapment in plasmid pUCD800 which carries the Bacillus subtilis sacB and sacR genes. The 1100-bp sequence contains 27-bp inverted repeats with 4 bp mismatch and has one long open reading frame, spanning 92.1% of the entire IS. The deduced 338 amino-acid sequence demonstrated homology (varying from 65% to 78% similarity and 36-67% identity) to transposases encoded by the IS30 family of IS elements. Comparison of four different IS-sacB junction sequences showed that IS1394 generated 3-bp direct repeats of target DNA upon insertion. IS1394 is present in at least 10 copies in the P25X genome but none was detected in its endogenous plasmid pRA2. Hybridization experiments revealed that the distribution of IS1394 is limited to closely related strains, being present in three copies in Pseudomonas putida NCIB 9869 (P35X) and two copies in Pseudomonas alcaligenes ATCC type strain (ATCC 14904).

Base Sequence↗

PubMed2006

In vitro transposon mutagenesis of an equine herpesvirus 1 genome cloned as a bacterial artificial chromosome.

The 150-kbp genome of the alphaherpesvirus equine herpesvirus 1 (EHV-1) strain HVS25A was cloned as a bacterial artificial chromosome (EHV-1 BAC), with mini F plasmid sequences inserted between genes 62 and 63. Transfection of EHV-1 BAC DNA purified from E. coli gave rise to progeny virus that had a similar growth rate and yield in mammalian cell culture to those of parental wild-type EHV-1. Using in vitro mutagenesis with a Mu transposon, a large library of EHV-1 BAC mutants was generated, and sequence analysis indicated that insertions were dispersed randomly across the EHV-1 genome. Following transfections of a pilot sample of mutant EHV-1 BAC DNAs into mammalian cells, no CPE was observable by light microscopy for mutants carrying insertions in genes for the major capsid protein, large tegument protein, glycoprotein K, catalytic subunit of DNA polymerase, or single-stranded DNA-binding protein. Mutants that were able to produce CPE similar to wild-type EHV-1 included those with interruptions in ORFs of several tegument proteins. Analysis of several glycoprotein gene mutants indicated that those carrying insertions near the start of genes encoding glycoproteins E and I were viable, but showed markedly diminished plaque areas. These results were supported by confocal microscopy of transfected or infected cultures. Electron microscopy of cells infected with a gE mutant revealed accumulations of particles within cytoplasmic vesicles, consistent with a partial obstruction of maturation. The transposon library is a resource for comprehensive functional analysis of the HVS25A genome, with multiple mutants available in any of the predicted genes of EHV-1.

Animals↗

PubMed1999

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110.

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.

Amino Acid Sequence↗

PubMed1996

Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion.

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains are generated in cattle persistently infected with noncytopathogenic (noncp) BVDV.cp BVDV strains are considered crucial for the development of fatal mucosal disease. Comparative analysis of cp and noncp BVDV strains isolated from one animal suffering from mucosal disease revealed that the genomes of the cp BVDV strain (CP7) and the corresponding noncp BVDV strain (NCP7) are highly homologous. However, only the genome of CP7 contains an insertion of 27 nucleotides in the NS2 coding region. The inserted sequence represents a duplication of bases 4064 to 4090 of the viral genome, located between the formerly neighboring nucleotides 4353 and 4354. Parts of the viral polyproteins of CP7 and NCP7 were expressed in the T7 vaccinia virus system. These studies revealed that the insertion identified in the CP7 genome is necessary and sufficient for the induction of NS2-3 cleavage. Since the expression of NS3 is strictly correlated to cp BVDV, the insertion identified in the genome of BVDV CP7 represents most likely the relevant mutation leading to the evolvement of CP7 from NCP7.

Animals↗

PubMed1992

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.

Bacteroides fragilis↗

PubMed2003

Prevalence and clinical implications of insertions in the HIV-1 p6Gag N-terminal region in drug-naive individuals initiating antiretroviral therapy.

We assessed the prevalence and clinical impact of insertions within the HIV-1 p6Gag proline-rich (PTAP) region on initial antiretroviral therapy response in 461 HIV-infected, drug-naive individuals initiating therapy in British Columbia, Canada between June 1996 and August 1998. HIV p6Gag insertions were detected by nested RT-PCR of extracted patient plasma followed by direct DNA sequencing. Insertions were observed in 70 of 423 successfully genotyped samples (16.5%). HIV p6Gag insertions were significantly associated with a lower baseline CD4 cell count (P<<0.05) and the presence of basic amino acids at key positions in the HIV envelope V3 loop linked to a syncytium-inducing phenotype (P<<0.05). After adjusting for baseline factors, no effect of HIV p6Gag insertions was observed on time to virological success (pVL < or = 500 copies/ml), virological failure (subsequent confirmed pVL > or = 500 copies/ml) or immunological failure (confirmed CD4 count below baseline), as evaluated by Kaplan-Meier methods and Cox proportional hazard regression (P>0.1). The data suggest that HIV p6Gag insertions are not exclusively related to drug resistance and may not influence response to antiretroviral therapy, but may be linked to sequence variations in the HIV envelope.

Adult↗

PubMed1998

Molecular cloning of human ABPL, an actin-binding protein homologue.

Based on two partial cDNA sequences, a full-length cDNA sequence for an actin-binding like protein previously named ABPL has been isolated and characterized. ABPL is homologous to the human actin-binding proteins ABP-280 and ABP-278. The predicted sequence for ABPL is 2,705 amino acids in length with a calculated molecular mass of 289 kDa. It contains an amino terminal actin-binding domain followed by 24 tandem repeats of approximately 96 amino acids. Two hinge regions, Hinge I and Hinge II, were located prior to repeats 16 and 24, respectively. An isoform of ABPL lacking Hinge I, with a calculated molecular mass of 286 kDa, was also identified by the reverse transcriptase PCR (RT-PCR) method. A comparison with genomic sequences indicated the isoform resulted from alternative RNA splicing. ABPL has a unique insertion sequence of 82 amino acids in repeat 20 that was not present in the other two homologues and has a tissue distribution that was also different from the other two homologues.

Actins↗