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PubMed1992

DNA sequence of IS91 and identification of the transposase gene.

IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.

Amino Acid Sequence↗

PubMed1998

FKBP-type peptidyl-prolyl cis-trans isomerase from a sulfur-dependent hyperthermophilic archaeon, Thermococcus sp. KS-1.

A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17kDa and the other 27kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC50 of 7 microM. This enzyme showed high kinetic stability with a half-life of 40 min at 100 degrees C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.

Amino Acid Sequence↗

PubMed2003

Chromosomal integration of a cephalosporinase gene from Acinetobacter baumannii into Oligella urethralis as a source of acquired resistance to beta-lactams.

Clinical Oligella urethralis isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into Escherichia coli and expression experiments gave recombinant clones expressing either an AmpC beta-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing beta-lactamase-type phenotype of resistance. The AmpC beta-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of Acinetobacter baumannii. A 820-bp insertion sequence element, ISOur1, belonging to the IS6 family of insertion sequence elements, was identified immediately upstream of bla(ABA-1), providing a -35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing beta-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of CeuI fragment I-restricted DNA fragments of O. urethralis COH-1 with bla(ABA-1)-, bla(CARB-8)-, and 16S rRNA-specific probes indicated the chromosomal integration of the beta-lactamase genes. PCR and hybridization experiments failed to detect bla(CARB-8)- and bla(ABA-1)-like genes in three O. urethralis reference strains, indicating that the beta-lactamase genes identified were the source of acquired resistance in O. urethralis COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring beta-lactamase.

Acinetobacter baumannii↗

PubMed1997

Subcellular localization of the MEN, MLL/MEN and truncated MLL proteins expressed in leukemic cells carrying the t(11;19)(q23;p13.1) translocation.

The t(11;19)(q23;p13.1) translocation is exclusively associated with myeloid leukemias. Previously, we cloned several species of MLL/MEN chimeric cDNAs in a patient with myeloid leukemia carrying the t(11;19)(q23;p13.1) translocation. The MEN sequence directly followed the 5' region of MLL cDNA in some species and otherwise there presented an inserted sequence of 120 bp between the MLL and MEN sequences in others. Because the insertion sequence contained an in-frame termination codon, they coded only for the NH2-terminal part of MLL (truncated MLL). We also cloned the normal MEN cDNA in full-length with a cDNA library derived from K562 cells. We expressed the normal MEN, MLL/MEN chimeric and truncated MLL proteins in COS7 cells with the corresponding cDNAs and detected them with antibodies raised against the MEN and MLL peptides. Immunostaining and subcellular fractionation showed nuclear localization of all these proteins. These findings suggested that MLL/MEN chimeric cDNAs were actually translated into both MLL/MEN fusion and truncated MLL proteins and that they were localized in the nucleus of leukemic cells. Recently, Conaway et al. reported that MEN is an RNA polymerase II elongation factor. The leukemogenesis by the t(11;19)(q23;p13.1) translocation may have resulted from the alteration of transcription regulation induced by the MLL/MEN fusion protein and/or the truncated MLL protein.

Animals↗

PubMed1991

IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting.

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.

Autoradiography↗

PubMed1986

Biologic activities of nonenzymatic thrombin: elucidation of a macrophage interactive domain.

Our studies to date have clearly demonstrated the existence of a unique cell interactive exosite region in thrombin, which mediates specific biologic effects on cells of monocytic-macrophage lineage. These findings are of potential physiologic significance, since even proteolytically degraded forms of thrombin (beta- and gamma-thrombin) or fragments of thrombin arising due to breakdown of thrombin-thrombomodulin complexes are potentially biologically active and are not subject to inhibition by inhibitors such as antithrombin III. Such degraded thrombin forms and proteolytic fragments are likely to be present at sites of inflammation and wound repair where they may be important modulators of macrophage-monocyte responsiveness. Importantly, as emphasized in this communication, the chemotactic and growth-promoting properties of this site, although overlapping, are clearly dissociable. It is apparent that the chemotactic effects require the presence of most, if not all, of the 338-400 sequence, whereas the mitogenic effects are mediated exclusively through the so-called loop B insertion sequence that is a unique feature of thrombin. Although there are ample precedents for differing functional domains within discrete proteins and the presence of hormonelike regions in proteins (such as fibronectin and immunoglobulin G) has been documented, it is fascinating to speculate how functionally unique regions such as this can arise. Data from Craik and associates suggest that unique amino acid sequences in proteins originate from gene insertions at intron-exon junctions and are characteristically surface expressed. These insertion sequences give rise to unique structural and functional features that characterize the particular protein, and also serve to differentiate it from other related members within its family.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Double cross-over gene replacement within the sec 7 domain of a GDP-GTP exchange factor from Plasmodium falciparum allows the generation of a transgenic brefeldin A-resistant parasite line.

High molecular weight ADP ribosylation factor GDP-GTP exchange factors (ARF-GEF) play an essential role in the formation of COP I coated transport vesicles and are characterized by a structurally and functionally conserved sec 7 domain. The genome of the malaria parasite Plasmodium falciparum encodes a single ARF-GEF that contains an unusual sec 7 domain. In comparison to the sec 7 domain of other eukaryotes, the plasmodial sec 7 domain is characterized by an insertion sequence of 146 amino acids that disrupt helices essential for the GDP-GTP exchange activity of the protein. In a previous study we have shown a correlation between a methionine to isoleucine exchange in helix H of the sec 7 domain and resistance to brefeldin A in a parasite line generated by drug selection. Here we have transfected brefeldin A sensitive parasites with plasmid constructs containing the sec 7 domain of the resistant line either with or without the insertion sequence. Transfection with sec 7 sequences including the insertion resulted in brefeldin A resistant parasites in which double cross-over recombination had replaced the endogenous sec 7 sequences with the transgenic sequences. Thus, the point mutation in helix H is sufficient to confer brefeldin A resistance in P. falciparum. Transfections using constructs lacking the insertion did not result in resistant parasites. Gene replacement by targeted double cross-over recombination is a rare event in P. falciparum. This approach has taken advantage of the fact that the successful integration of the transgene results in a drug selectable phenotype. We anticipate that the strategy described here will be useful for the identification of mutations within target genes that have the potential to confer increased drug resistance.

ADP-Ribosylation Factors↗

PubMed1994

Transposition of IST2 in Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IST2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IST2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IST2 is a mobile insertion sequence.

Amino Acid Sequence↗

PubMed2005

Pyrrolysine and selenocysteine use dissimilar decoding strategies.

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies.

Amino Acid Sequence↗

PubMed1997

High-resolution functional mapping of a cloned gene by genetic footprinting.

We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization. The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest. This mutant library is selected for gene function en masse. DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed. The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility. For the whole library, this results in a ladder of bands, each band representing a specific mutation. Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection. The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function. Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants. This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences.

Chromosome Mapping↗

PubMed1988

In vivo gene transfer systems and transposons.

The continued development of our understanding and application of the in vivo gene transfer systems, transduction and conjugation, and the more recent use of transposons in lactic acid bacteria is reviewed. The discovery of transduction and its use as a tool for genetic analysis is described. The history of the use of conjugation in lactic acid bacteria is outlined, but more detailed discussion is reserved for in-depth analysis of the conjugation system associated with the lactose plasmids of Streptococcus lactis strains ML3 and C2. This system is notable for an unusual cell aggregation phenotype associated with variants of lactose plasmids capable of high frequency transfer and the complex DNA interactions associated with this property. Recent advances in the use of wide host range conjugation systems, such as that of plasmid pAM beta 1 are described, including the mobilisation of vectors by cointegrate formation and subsequent segregation after transfer. The successful exploitation of conjugation for the construction of bacteriophage-resistant starter cultures is highlighted. A description of transposable genetic elements in the lactic acid bacteria, both insertion sequences and transposons, puts emphasis on the elegant analysis of insertion sequence ISL1 in Lactobacillus casei and on the exploitation of the conjugative transposons Tn916 and Tn919. The latter is especially important for providing a technology to initiate analysis of the bacterial chromosome of the lactic acid bacteria.

Conjugation, Genetic↗

PubMed1987

Reversible interconversion between primitive endoderm- and parietal endoderm-like F9 cells demonstrated by mRNAs expression.

The differentiation of retinoic acid-treated F9 cells (primitive endoderm-like F9 cells) into parietal endoderm-like F9 cells induced by dibutyryl cAMP was studied as a culture model of the morphogenesis of early mouse embryo. For this purpose, 6 cDNA clones coding for mRNAs specifically expressed in parietal endoderm-like F9 cells were selected. Northern hybridization of RNA extracted from variously treated F9 cells to nick-translated plasmid DNA of these clones demonstrated the reversible expression of many mRNAs depending on the presence of dibutyryl cAMP in the culture medium. This result suggested that the differentiated state of parietal endoderm, which is formed from primitive endoderm at a position adjacent to the trophectoderm in mouse embryo, can be reversed if the local signal is removed. One of the selected clones, pLAM, hybridized to an mRNA of 6.3 kb and selected mRNA producing a laminin B subunit in an in vitro translation system. This clone has an inserted sequence of 3.1 kb. Among the restriction sites in this sequence, six were consistent with those in a 1.7 kb inserted sequence of pPE 49 and pPE 386, which were isolated by Barlow et al. as laminin B1 clones. An XbaI site found in both pPE 49 and pPE 386 was, however, not found at the corresponding position of pLAM. Dot hybridization of RNA with pLAM showed that expression of laminin B in F9 cells is stimulated more than 100-fold during differentiation of F9 stem cells into parietal endoderm-like F9 cells.

Animals↗

PubMed1994

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Amino Acid Sequence↗

PubMed2006

LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease: mutation detection bias and multiple mechanisms of target gene disruption.

LINE-1 (L1) elements are the most abundant autonomous non-LTR retrotransposons in the human genome. Having recently performed a meta-analysis of L1 endonuclease-mediated retrotranspositional events causing human genetic disease, we have extended this study by focusing on two key issues, namely, mutation detection bias and the multiplicity of mechanisms of target gene disruption. Our analysis suggests that whereas an ascertainment bias may have generally militated against the detection of autosomal L1-mediated insertions, autosomal L1 direct insertions could have been disproportionately overlooked owing to their unusually large size. Our analysis has also indicated that the mechanisms underlying the functional disruption of target genes by L1-mediated retrotranspositional events are likely to be dependent on several different factors such as the type of insertion (L1 direct, L1 trans-driven Alu, or SVA), the precise locations of the inserted sequences within the target gene regions, the length of the inserted sequences, and possibly also their orientation.

Journal Article↗

PubMed1992

Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines.

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.

Amino Acid Sequence↗

PubMed1991

A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis PCC6803.

A clone that transforms the RKa mutant of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport to the wild-type phenotype was isolated from a cyanobacterial genomic library. The clone contained an 11.8-kilobase-pair DNA insert. Sequencing of the insert DNA in the region of the mutation in RKa revealed an open reading frame (designated as ndhB), which showed extensive amino acid sequence homology to the subunit-2 genes of NADH dehydrogenase (EC 1.6.99.3) (ndhB) of chloroplasts and mitochondria. The homology was much stronger with the chloroplast genes. Sequence analysis of the ndhB gene of RKa mutant revealed a G----A substitution that results in a Gly----Asp substitution in the deduced amino acid. A defined mutant (M55), constructed by inactivating the ndhB gene in wild-type Synechocystis, required high CO2 conditions for growth and was unable to transport CO2 and HCO3- into the intracellular Ci pool. The results indicate that the ndhB gene is required for Ci transport. Dark respiration was also depressed by the inactivation of the ndhB gene. A possible role of the ndhB gene product in the energization of Ci transport is discussed.

Amino Acid Sequence↗

PubMed1988

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.

Animals↗

PubMed1988

Activation of silent genes by transposons Tn5 and Tn10.

The presence of transposons Tn10 or Tn5 in the genome increases the frequency with which a silent (promoter-less) gene (hisD) is mutationally activated. The activation frequency is increased 5-25-fold by Tn10 and 30-90-fold by Tn5. Activation of the hisD gene is achieved by transposition of the entire transposon or one of its flanking insertion sequences to a region just upstream of the silent gene, between a Rho-dependent termination site in the adjacent hisG gene and the hisD gene. For both Tn5 and Tn10 the component insertion sequences were found to transpose much more frequently than the entire composite element. Transposons Tn5 and Tn10 have previously been shown to carry promoters which direct transcripts into sequences adjacent to their insertion sites.

Alcohol Oxidoreductases↗