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PubMed2003

Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus.

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).

Base Sequence↗

PubMed1992

Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.

Base Sequence↗

PubMed1995

IS292: a novel insertion element from Agrobacterium.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Amino Acid Sequence↗

PubMed1999

Detection of a normally rare transcript in propionic acidemia patients with mRNA destabilizing mutations in the PCCA gene.

Propionic acidemia is an autosomal recessive disorder caused by a deficiency in the mitochondrial enzyme propionyl-CoA carboxylase (PCC). PCC is composed of two subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. We analyzed mutations of the PCCA gene using patients' fibroblasts diagnosed with alpha subunit deficiency. By RT-PCR, four of 12 cell lines examined appeared to have a larger transcript present at a level comparable with that of the expected normal species. Sequencing of the larger transcriptrevealed an 84 bp insertion at nt 1209 of the codingsequence. Its incorporation in the transcript results in translation termination due to the presence of two in-frame stop codons. The 84 bp insertion was found to originate from the intron between nt 1209 and 1210. Consensus splice donor and acceptor sites were found at the 3'- and 5'-ends of the insertion, respectively. The insertion was also found in the remaining eight cell lines as well as in normal cells, but at a muchreduced level compared with the normal lengthsequence. Mutation analysis of the four cell lines showing seemingly elevated levels of the insertion sequence revealed one nonsense mutation (R288X), two frameshift deletions (700del5 and 1115del4) and one splice mutation (1671IVS+5G-->C) as expressed alleles. We conclude that the common characteristic of the four cell lines is that they contain mRNA destabilizing mutations that reduce the mRNA level of the normal length sequence. Consequently, the low levels of cryptic mRNAs become detectable at a level similar to that of the residual level of the normal length mRNA. We suggest that screening for an increased proportion of the 84 bp insertion by RT-PCR can be used as a rapid assay for RNA destabilizing mutations. Our results suggest caution in associating such mutations with aberrant mRNA species, such as cryptic splice products, which may instead be part of the 'background noise' of the splicing machinery.

Base Sequence↗

PubMed2001

RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.

Animals↗

PubMed1996

Neuron-specific alternative splicing of nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron sequence.

In addition to the ubiquitously expressed form of nonmuscle myosin II heavy chain-B (MHC-B), the existence of a neuron-specific MHC-B isoform, which contains a 30-nucleotide inserted sequence near the ATP binding region, has been reported (Takahashi, M., Kawamoto, S., and Adelstein, R. S.(1992) J. Biol. Chem. 267, 17864-17871). In this study, the genomic location of the neuron-specific inserted 30-nucleotide sequence found in the cDNA is determined to be a single cassette type exon, N30, in the human nonmuscle MHC-B gene. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neuronal cells and being regulated during cell differentiation. Expression of a minigene construct that contains the alternative exon N30 along with the flanking introns and exons was studied in human neuronal retinoblastoma Y79 cells. Inclusion of the N30 exon in the mRNA from the transfected minigene occurs in differentiated Y79 cells that have been treated with butyrate but not in the undifferentiated Y79 cells and non-neuronal cell lines. Systematic deletion and mutation analysis of the minigene construct established that neuron-specific N30 exon recognition requires a cis-acting RNA sequence located approximately 1.5 kilobases downstream of the N30 exon.

Alternative Splicing↗

PubMed2005

[Insertion of vector sequences in the genome of transgenic plants].

Insertion of vector sequences of different lengths in the genome of transgenic tobacco and carrot plants occurring at a frequency of 35.2% was shown. No significant differences in insertion frequencies between the two species were observed. Integrated fragments of the vector DNA were stably inherited in the following generation resulted from self-pollination of original transformants.

Daucus carota↗

PubMed2004

A large MSH2 Alu insertion mutation causes HNPCC in a German kindred.

Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is an autosomal, dominantly inherited disease accounting for about 1%-5% of all colorectal cancer cases. HNPCC predisposition is caused by germline mutations in at least five genes coding for DNA mismatch repair (MMR) proteins. More than 400 MMR gene mutations have been identified in HNPCC patients. About 90% of mutations affect the MLH1 and MSH2 genes. The mutational spectrum mainly includes point mutations and small deletions or insertions. Here, we report a large 184 base-pair Alu insertion mutation in exon 6 of the MSH2 gene in a German HNPCC family. The inserted sequence contains repetitive Alu sequence elements that present the highest homology with the old Alu J subfamily. The Alu J insertion was most likely derived from Alu-mediated recombination, since Alu J elements have been found close to the insertion site in adjacent introns, and since elements pivotal for Alu retrotransposition are missing. Our results suggest that the recombination event occurred at least one generation ago. This is the first report of an Alu insertion in the coding sequence of a MMR gene as the cause of HNPCC. Our data thus further extend the spectrum of MMR gene mutations causative for HNPCC.

Aged↗

PubMed1986

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells.

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase (aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the aprt locus, two mutant genes were cloned from lambda genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.

Animals↗

PubMed1997

The seventh copy of IS1 in Escherichia coli W3110 belongs to the IS1 A (IS1E) type which is the only IS1 type that transposes from chromosome to plasmids.

In the Escherichia coli K-12 chromosome, six copies of IS1 (IS1A - IS1F) have been identified and characterized. According to their nucleotide (nt) sequences, the six IS1 copies can be classified into four types, IS1A(IS1E), IS1B(IS1C), IS1D and IS1F type. Zuber and Schumann (1993) identified the seventh IS1 copy at 49.6 minutes on the E. coli W3110 genetic map. Unfortunately, only the end 21-bp sequence as well as the neighboring 120-bp E. coli sequence were reported. We therefore designed two oligonucleotide primers to specifically amplify the seventh IS1 copy by PCR. One primer is homologous to the first sixteen bases of the IRL sequence of IS1A(IS1E), IS1B(IS1C) and IS1D. The other primer is complementary to the eighteen bases of E. coli sequence adjacent to IRR of the seventh IS1 copy. An 800-bp PCR fragment was obtained and its nt sequence determined, revealing an identical nucleotide sequence to that of IS1A(IS1E). A plasmid system was then used to isolate insertion mutations caused by insertions of the chromosomal insertion sequences. Of the 142 plasmid insertion mutants isolated, thirty-eight were insertions of chromosomal IS5, ten were IS30, and ninety-four were IS1. Detailed restriction mapping indicates that all ninety-four plasmid IS1 insertions were insertions of IS1 of the IS1A(IS1E) type.

Chromosomes, Bacterial↗

PubMed1990

Insertion of IS4 into the tnpA gene of Tn3: sequencing of the insertion site.

Plasmid pMM237 lacks the deletion-forming activity associated with its Tn3 transposase-encoding gene (mpA) [Morita et al., J. Biochem. 101 (1987) 1253-1264]. Analysis of the plasmid DNA showed that pMM237 was generated by insertion of IS4 into the tnpA gene of the original plasmid pMM234. The insertion was accompanied by a direct 12-bp duplication at the target site. The nucleotide sequence around the target site shared only some of the characteristics of previously reported IS4 target sites [Habermann et al., Mol. Gen. Genet. 175 (1979) 369-373; Klaer et al., Mol. Gen. Genet. 181 (1981) 169-175; Georgopoulos et al., Gene 20 (1982) 83-90; Mayaux et al., Gene 30 (1984) 137-146].

Autoradiography↗

PubMed1984

Construction and applications of a highly transmissible murine retrovirus shuttle vector.

We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousands of bacterial transformants per 5 X 10(6) mammalian cells. The majority of plasmids recovered by all three protocols exhibited the proper structure and were as active as the parental vector in the generation of transmissible retrovirus genomes upon transfection of mammalian cells. One of the rescue methods, which relies on "onion skin" replication and excision of an integrated provirus from the host chromosome, enables facile recovery of the chromosomal site of proviral integration. The system was also used to generate, and then efficiently recover, a cDNA version of a genomic insert from the adenovirus E1A region.

Cell Transformation, Viral↗

PubMed2000

IS1294, a DNA element that transposes by RC transposition.

IS1294, found on the ColD-like resistance plasmid pUB2380, is IS91-like. It is an active 1.7-kb insertion sequence that lacks terminal inverted repeats, displays insertion-site specificity, and does not generate direct repeats of the target site. The element has one large open reading frame, tnp(1294), encoding a transposase of 351 amino acids, related to members of the REP family of replication proteins used by RC-plasmids of gram-positive bacteria. IS1294 transposes using rolling-circle replication, initiated at one end of the element, oriIS, and terminated at the other, terIS. oriIS and terIS are highly conserved among like IS elements. oriIS resembles the leading strand replication origins of RC-plasmids; terIS resembles a rho-independent transcription terminator. IS1294 mediates not only its own transposition, but also sequences adjacent to terIS. A transposition model for IS1294 and related elements, involving rolling-circle replication and single-strand DNA intermediates, is presented.

Amino Acid Sequence↗

PubMed2006

Isolation and expression of the lysis genes of Actinomyces naeslundii phage Av-1.

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective lambda holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.

Actinomyces↗

PubMed1993

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin.

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from alpha-carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an alpha-helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core beta-barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isoleucine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressed in vivo.

Amino Acid Sequence↗

PubMed1993

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii.

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a 'yellow-in-the-dark' phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.

Algal Proteins↗

PubMed1999

Disruption of coding regions by IS6110 insertion in Mycobacterium tuberculosis.

SETTING: The insertion sequence IS6110 is widely used as a DNA fingerprinting probe for the classification of Mycobacterium tuberculosis strains. This study has focused on the characterization of regions disrupted by insertion of the IS6110 element. OBJECTIVE: To characterize IS6110 insertion loci in clinical isolates of M. tuberculosis, in terms of their genomic location and genetic identity, to ascertain whether IS6110 transposition could be a mechanism driving phenotypic change. DESIGN: Thirty-three IS6110 insertion loci were cloned from 8 clinical isolates of M. tuberculosis. Clones representing DR locus insertions were identified by hybridization (n = 4), and all other clones were characterized by DNA sequencing (n = 29). The sequence data was analyzed in conjunction with that of 43 other insertion loci identified in published literature and DNA sequence databases. RESULTS: The 76 sequences analyzed represented 66 unique insertion loci (including 9 unique insertions into the ipl locus). When mapped to the H37Rv genome, the majority of unique insertion loci demonstrated disruption of coding regions by IS6110 (n = 42; including the ipl insertions), while the remainder either occurred within intergenic regions (n = 17), or could not be mapped to the H37Rv genome sequence (n = 7). Mapping of the insertion loci reveals distribution throughout the chromosome, with isolated preferential insertion loci. CONCLUSIONS: This study has demonstrated the occurrence of 66 unique IS6110 insertion loci dispersed throughout the M. tuberculosis genome, with an unexpectedly high incidence of IS6110 insertions occurring within coding regions. However, the IS6110-mediated coding region disruptions identified here may only have limited impact on phenotype, as most of the coding regions disrupted are members of multiple gene families. Disruption of individual members of a family of genes may have no effect on phenotype or could have a minor or major impact, depending on the specificity and activity of the encoded protein.

DNA Fingerprinting↗

PubMed1996

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder.

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50% in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms.

Adenosine Triphosphate↗