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PubMed1989

Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila.

The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development. We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA. In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA. Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested. Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly. This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines. One of the 26S rRNA mutants failed to generate transformants, but the other did. These transformants grew normally, and produced 26S rRNA containing the inserted sequence. A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA. This study reveals that some rRNA sequences can be altered without significantly affecting cell growth.

Animals↗
PubMed1988

Composite IS1-tetracycline resistance elements in aerobactin-encoding FIme plasmids from epidemic Salmonella wien.

Class B tetracycline resistance determinants have been identified in two aerobactin-encoding FIme plasmids representative of those isolated from epidemic Salmonella wien. Genetic data, restriction enzyme analysis of recombinant and mutant plasmids, and Southern blot hybridizations indicate that in both plasmids the class B determinant so far found and described only on Tn10-like transposons is part of a different genetic element. This composite insertion sequence element is about 7 kilobases long and has copies of insertion sequence IS1 at the ends.

Blotting, Southern↗
PubMed2000

Dysregulated expression of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus-prone mice.

The Cd22 gene encodes a B cell-specific adhesion molecule that modulates B cell Ag receptor-mediated signal transduction, and is allelic to a lupus-susceptibility locus in New Zealand White (NZW) mice. In this study, we show that, in addition to the wild-type transcripts, NZW (Cd22a) mice synthesize aberrant CD22 mRNAs that contain approximately 20-120 nucleotide insertions upstream of the coding region between exons 2 and 3, and/or approximately 100-190 nucleotide deletions of exon 4. Sequence analysis revealed that these aberrant mRNA species arose by alternative splicing due to the presence in the NZW strain of a 794-bp sequence insertion in the second intron, containing a cluster of short interspersed nucleotide elements. Both the presence of sequence insertion and aberrantly spliced mRNAs were specific to mice bearing the Cd22a and Cd22c alleles. Up-regulation of CD22 expression after LPS activation appeared impaired in Cd22a spleen cells (twice lower than in Cd22b B cells). Furthermore, we show that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, markedly promotes the production of IgG anti-DNA autoantibodies in C57BL/6 (Cd22b) mice bearing the Y chromosome-linked autoimmune acceleration gene, Yaa. Taken together, these results suggest that a lower up-regulation of CD22 on activated B cells (resulting from Cd22 gene anomaly in Cd22a mice or from CD22 heterozygosity in mutants obtained by gene targeting) is implicated in autoantibody production, providing support for Cd22a as a possible candidate allele contributing to lupus susceptibility.

5' Untranslated Regions↗
PubMed1981

Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar.

Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient.

Bacteriophage lambda↗
PubMed2000

Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements.

As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations. In both populations, multiple IS-associated mutations arose that then went to fixation. We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations. All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions. Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU). These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation.

Biological Evolution↗
PubMed2000

pT3.2I, the smallest plasmid of Thiobacillus T3.2.

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3. 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.

Amino Acid Sequence↗
PubMed1986

Molecular and biological properties of BK virus-IR, a BK virus variant isolated from a human tumor.

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.

Adenoma, Islet Cell↗
PubMed1991

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India.

Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5' termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.

Base Sequence↗
PubMed2006

Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis.

OBJECTIVES: A Salmonella enterica serovar Infantis isolate of avian origin was investigated for the presence of the gene qnrS, its transferability and its association with other resistance genes. METHODS: The Salmonella Infantis isolate was investigated for its susceptibility to antimicrobial agents and its plasmid content. Hybridization experiments and PCR assays were performed to identify the resistance genes while transformation and conjugation studies were conducted to show their transferability. The quinolone resistance-determining regions of the genes gyrA, gyrB, parC and parE were sequenced. Moreover, extended sequence analysis was performed to gain insight into the structure and organization of the qnrS gene area. RESULTS: The Salmonella Infantis isolate exhibited the Asp87-->Tyr87 mutation in gyrA, but no resistance-mediating mutations in the other target genes. It carried a conjugative plasmid, pINF5, on which a qnrS gene was detected in close proximity to a Tn3-like, blaTEM-1-carrying transposon. Homology to the qnrS-carrying plasmid pAH0376 of Shigella flexneri was limited to the Tn3-qnrS region. Sequence analysis of an approximately 13.4 kb region of pINF5 identified truncated insertion sequences of types IS26 and IS2 as well as an internal segment of the CS12 fimbrial gene cluster of Escherichia coli up- and downstream of the qnrS gene. CONCLUSIONS: This is to the best of our knowledge the first report of a qnrS gene in a Salmonella isolate. The analysis of the regions flanking the qnrS gene suggested that this region developed in multiple steps that included not only the integration of insertion sequences and a Tn3-like transposon, but also interplasmid recombination events.

Ampicillin↗
PubMed2005

Tn5393d, a complex Tn5393 derivative carrying the PER-1 extended-spectrum beta-lactamase gene and other resistance determinants.

In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum beta-lactamase, the bla(PER-1) gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the bla(PER-1) gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the bla(PER-1) gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the bla(PER-1) genes, respectively. The putative composite transposon carrying bla(PER-1), named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the bla(PER-1) gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.

Alcaligenes faecalis↗
PubMed1990

Molecular biology of Crohn's disease mycobacteria.

A Glasgow surgeon, T.K. Dalziel, published a detailed description of chronic enteritis in humans in 1913. He proposed that the disease was caused by the same organisms as those responsible for chronic enteritis, Johne's disease, in animals described a few years earlier (1895). Dalziel's dilemma was that he could see acid-fast bacilli in the diseased animal tissues but not in the diseased human tissues. Little real progress in the medical understanding of the causes of chronic enteritis in humans occurred over the next half a century or more. From 1978, a decade of research in many laboratories using improved methods for the culture of environmental mycobacteria showed that these could be grown in bacillary form from about one in five cases of Crohn's disease, from the same proportion of cases of ulcerative colitis, and from about one in ten control tissues. Spheroplasts were grown from two in five cases of Crohn's disease, one in five cases of ulcerative colitis, and rarely from control tissues. The nature of these agents was often uncertain. We describe work which began in 1985 and led rapidly to the identification of IS900, a DNA repetitive element in an uncharacterized Crohn's disease mycobacterial isolate. With other isolates, these were then shown by DNA fingerprinting to be indistinguishable from Mycobacterium paratuberculosis, Johne's bacillus. Similar techniques also demonstrated the wood-pigeon strain of M. avium in some Crohn's disease cultures. This bacillus can also cause chronic enteritis in calves. IS900 is the first of a family of unusual DNA insertion sequences which extend widely throughout environmental mycobacteria. Use of assays based on PCR amplification of highly specific DNA sequences from these insertional elements, and recombinant and synthetic peptides from their predicted proteins, will revolutionize the detection and characterization of these agents. These methods, applied to animal, human and environmental samples, will indicate new ways for the prevention and treatment of chronic enteritis, as well as other disorders associated with infections by environmental mycobacteria.

Actinomycetales↗
PubMed2000

Characterization of IS666, a newly described insertion element of Mycobacterium avium.

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.

Amino Acid Sequence↗
PubMed1989

Genetic variation occurring on the genome of an in vitro insertion mutant of poliovirus type 1.

An insertion sequence of 72 nucleotides prepared from a polylinker sequence of plasmid pUC18 was introduced at nucleotide position 702 of the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of poliovirus type 1 by using an infectious cDNA clone of the virus strain. The insertion mutant thus obtained showed a small-plaque phenotype compared with that of the parent virus. Apparent revertants (large-plaque variants) were easily generated from the insertion mutant. Nucleotide sequence analysis was performed on the revertant genomes to determine the mutation(s) by which the plaque size of the parent virus was regained. Some large-plaque variants lacked genomic sequences including all or a part of the insertion sequence. A computer-aided search for secondary structures with respect to the deletion sites detected possible supporting sequences which provided fairly stable secondary structures at the deletion sites. This result was consistent with our supporting sequence-loop model which had been proposed as a new copy-choice model for the generation of genetic rearrangements occurring on single-stranded RNA genomes (S. Kuge, I. Saito, and A. Nomoto, J. Mol. Biol. 192:473-487, 1986). The other large-plaque variants had point mutations at any one of three positions of an AUG existing in the insertion sequence. A small-plaque phenotype was observed when an AUG codon was inserted in frame or out of frame with regard to the initiation site of viral polyprotein synthesis. Our data strongly suggest that an AUG sequence in this genome region is deleterious for efficient poliovirus replication.

Chromosome Deletion↗
PubMed1989

Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles.

Partial acid cleavage, comparative HPLC tryptic peptide mapping and amino acid sequencing of the C1 and C2 proteins of HeLa heterogeneous nuclear ribonucleoprotein (hnRNP) particles demonstrate that proteins C1 and C2 differ in primary structure by the presence of a 13 amino acid insert sequence in C2. This C2 insert sequence occurs after either glycine 106 or serine 107 in C1. The additional 13 amino acids that are present in C2 account for the observed molecular weight difference between the C1 and C2 hnRNP proteins on SDS polyacrylamide gel electrophoresis. Because C1 and C2 appear identical except for the 13 residue insert and because the 3' and 5' untranslated regions of the corresponding mRNAs also appear to be the same (Swanson et al., Mol. Cell. Biol. 7: 1731-1739), it is possible that both polypeptides are produced from a single transcription unit through an alternative splicing mechanism.

Amino Acid Sequence↗
PubMed2006

Prospective study of fetal DNA in serum and disease activity during pregnancy in women with inflammatory arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) usually improves during pregnancy and recurs postpartum. Fetal cells and cell-free DNA reach the maternal circulation during normal pregnancy. The present study investigated dynamic changes in levels of fetal DNA in serum from women with RA and inflammatory arthritis during and after pregnancy to test the hypothesis that the levels of circulating fetal DNA correlate with arthritis improvement. METHODS: Twenty-five pregnant patients were prospectively studied. A real-time quantitative polymerase chain reaction panel targeting unshared, paternally transmitted HLA sequences, a Y chromosome-specific sequence, or an insertion sequence within the glutathione S-transferase M1 gene was used to measure cell-free fetal DNA. Results were expressed as fetal genomic equivalents per milliliter (gE/ml) of maternal serum. Physical examinations were conducted during and after pregnancy. RESULTS: Levels of fetal DNA in women with improvement in or remission of arthritis were higher than those in women with active disease, especially in the third trimester. Overall, an inverse relationship between serum fetal DNA levels and disease activity was observed (P < 0.001). Serum fetal DNA increased with advancing gestation, reaching median levels of 24 gE/ml (range 0-334), 61 gE/ml (range 0-689), and 199 gE/ml (range 0-2,576) in the first, second, and third trimesters, respectively, with fetal DNA clearance observed postpartum. Arthritis improvement was initially noted in the first trimester for most patients, increased further or was sustained with advancing gestation, and was active postpartum. CONCLUSION: Changes in serum fetal DNA levels correlated with arthritis improvement during pregnancy and recurrence postpartum. Immunologic mechanisms by which pregnancy might modulate RA activity are described.

Adult↗
PubMed1978

Chloramphenicol resistance in Streptomyces coelicolor A3(2): possible involvement of a transposable element.

The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1- superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1-) it was never displaced by the immigrant chromosome even when the region facing chl+ was selected for. A fraction of the original Chl- mutants presented a requirement for arginine (ArgB-). A Chl- mutant gave rise spontaneously to ArgB- derivatives at high frequency. The same ArgB- requirement come out at high frequency among Chl- derivatives from a cross NFChl- x SCP1-Chl+ in which neither parent required arginine or produced spontaneously arginine-less derivatives. It is suggested that the Chl element is a "transposable element" (Tn) presumably associated with "insertion sequences" (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.

Arginine↗
PubMed2005

Nonhomologous recombination between the large unassigned region of the male and female mitochondrial genomes in the mussel, Mytilus trossulus.

Doubly uniparental inheritance of mtDNA (DUI) is commonly observed in several genera of bivalves. Under DUI, female offspring inherit mtDNA from their mothers, while male offspring inherit mtDNA from both parents but preferentially transmit the paternally inherited mtDNA to their sons. Several studies have shown that the female- and male-specific mtDNA lineages in blue mussels, Mytilus spp., vary by upward of 20% at the nucleotide level. In addition to high levels of nucleotide substitution, the present study observed substantial gender-based length polymorphism in the presumptive mitochondrial control region (=large unassigned region; LUR) of North American M. trossulus. In this species, female lineage LUR haplotypes are over 2 kb larger than male lineage LUR haplotypes. Analysis of sequence data for these length variants indicates that the F LUR haplotypes of North American M. trossulus contain sequences similar to the F lineage control region in the congeners M. edulis and M. galloprovincialis. Relative to the F LUR in the latter two species, however, the F lineage LUR haplotypes in M. trossulus contain two large sequence insertions, each nearly 1 kb in size. One of these insertions has high sequence similarity to the male lineage LUR of M. trossulus. The tandem arrangement of F and M control region sequences in the F lineage LUR of M. trossulus is most likely the result of nonhomologous recombination between the male and the female mitochondrial genomes in M. trossulus, a finding that has important implications regarding the transmission and evolution of blue mussel mitochondrial genomes.

Animals↗