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PubMed1990

Centromere structure and function in budding and fission yeasts.

Functional centromeric DNAs have now been isolated and characterized from both budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts. Artificial chromosomes containing these centromere DNA sequences segregate faithfully in both mitotic and meiotic cell divisions, but only in the parent organism. Structure-function analyses have revealed surprising fundamental differences between these two centromere classes. In the budding yeast centromeres, a 125-bp consensus DNA sequence contains all the information needed in cis to provide proper chromosome segregation. In contrast, the fission yeast centromeres each contain a long run (40 to 100 kb) of untranscribed repetitive DNA sequences arranged into a large inverted repeat, most of which is required for full centromere function. The fission yeast centromere-kinetochore appears to be a highly relevant experimental model for analysis of the mechanism of chromosome segregation in higher eukaryotes, in which the centromere regions often contain megabases of transcriptionally silent repetitive DNA sequences of unknown function.

Base Sequence↗
PubMed1989

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001.

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.

Amino Acid Sequence↗
PubMed1984

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium.

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage PhiH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pPhiHL, consisting of the invertible L segment of the phage genome which is, in PhiH2 and PhiH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.

Journal Article↗
PubMed1983

Genome fusion mediated by the site specific DNA inversion system of bacteriophage P1.

The genome of bacteriophage P1 contains a segment which is invertible by site specific recombination between sequences near the outside ends of the inverted repeats which flank it. Immediately adjacent to this C segment is the coding sequence for cin, the enzyme catalyzing inversion. We show that multicopy plasmids carrying cin and the sequences at which it acts (cix) can form dimers in the absence of the host recA function. Further, such plasmids can be cotransduced with P1 markers at high frequency from recA lysogens, indicating cointegration with the P1 genome. It is thus demonstrated that a system whose primary role is the inversion of a specific DNA segment can also mediate intermolecular recombination.

Bacterial Proteins↗
PubMed1980

Cytomegalovirus: genetic variation of viral genomes.

Genomic complexity of human CMV is one of the largest among various DNA viruses. With such genome complexity and widespread distribution, even a minimal frequency of mutation will be enough to create a complicated genetic heterogeneity in this virus. The virus genome is relatively stable during subcultivation in tissue culture. Variants with minor modification in restriction enzyme sites do gradually develop. These variants share substantial restriction fragment pattern homology with the parental virus. Virus DNA has a molecular weight of 150 X 10(6) daltons for a complete genome. The DNA contains 2 covalently linked L and S components which are separated by internal inverted repetitious sequences of both terminal-end sequences. L and S components are oriented invertably with 4 isomeric arrangements. No tandem repeated sequences have been found. Based on DNA restriction pattern analysis, we conclude that the majority of recurrent infections represent reactivation of existing latent viruses; however, reinfections by a new virus strain do occur occasionally. By studying virus strains isolated from the consecutive congenitally infected infants and strains from mothers and their congenitally infected offspring, we furthermore conclude that the latent virus in women is relatively stable genetically. Moreover, the virus strains after being transmitted to the offspring are still genetically similar to that of the mothers. As in vitro, spontaneous minor variations occur after the in vivo residence. In a long period of evolution the accumulation of minor variations might create great diversity with major similarity.

Blood Donors↗
PubMed2005

Neisseria gonorrhoeae FitA interacts with FitB to bind DNA through its ribbon-helix-helix motif.

The fit locus, encoding two proteins, FitA and FitB, was identified in a genetic screen for Neisseria gonorrhoeae determinants that affect trafficking across polarized epithelial cells. To better understand how the locus may control these activities, we have undertaken a biochemical analysis of FitA and FitB. FitA is a DNA-binding protein with a putative ribbon-helix-helix (RHH) motif. Purified FitA forms a homodimer that binds a 150 bp fit promoter sequence containing the translational start site. A putative beta strand mutant of FitA, FitA(R7A), is unable to bind this DNA, supporting further that FitA is a RHH protein. FitB interacts with FitA to form a 98 kDa complex. FitA/B binds DNA with a 38-fold higher affinity than the FitA homodimer. In DNase I footprint assays, FitA/B protects a 62-bp region within the fit promoter containing the predicted -10 sequence and an 8-bp inverted repeat, TGCTATCA-N(12)-TGATAGCA. FitA/B(His) is able to bind to either half-site alone with high affinity.

Amino Acid Sequence↗
PubMed1987

The sequence of the 3.3-kilobase repetitive element from Dipodomys ordii suggests a mechanism for its amplification and interspersion.

DNA from the kangaroo rat, Dipodomys ordii, contains a 3.3-kb, highly repeated sequence that is interspersed throughout the genome in small tandem clusters. One 3.3-kb unit has been cloned into pBR322 and the nucleotide sequence determined. The clone used was shown to be representative of the bulk of such sequences found in the genomic DNA. The sequence contains 10 homologous subunits each ca. 260 bp in length. Comparison of these to one another yielded a 258-bp consensus sequence containing a 35-bp terminal inverted repeat. Two unique stretches also occur. One of these contains a region that could serve as a promoter for RNA polymerase III; the other contains a sequence related to the ARS sequences of yeast. It is proposed that an ancestral sequence similar to the consensus sequence was amplified to 10 or more units, and that, subsequently, two other sequences were inserted. The properties of these insertions may have led to the dispersal of the sequence throughout the genome.

Animals↗
PubMed2001

In vitro processing of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus.

In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5' external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdom Crenarchaeota but apparently not in the Euryarchaeota. Furthermore, S. solfataricus pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and the Euryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5' or the 3' end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.

Base Sequence↗
PubMed1991

Characterization of two insertion sequences, IS701 and IS702, from the cyanobacterium Calothrix species PCC 7601.

We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.

Amino Acid Sequence↗
PubMed1988

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.

Base Sequence↗
PubMed1987

Binding of multiple nuclear factors to the 5' upstream regulatory element of the murine major histocompatibility class I gene.

Transcription of mouse major histocompatibility complex class I genes is controlled by the conserved class I regulatory element (CRE) in the 5' flanking region. The CRE, approximately 40 base pairs long, acts as a negative control element in undifferentiated F9 embryonal carcinoma cells which do not express the major histocompatibility complex genes. The same element, however, acts as a positive control element in cells expressing the genes at high levels. To investigate the molecular basis of the regulatory role of the CRE, we studied the binding of nuclear proteins to the CRE of the H-2Ld gene by gel mobility shift and methylation interference experiments. Nuclear extracts from L fibroblasts and LH8 T lymphocytes revealed three distinct factors that bind discrete sequences within the CRE. The three sequences correspond to the inverted and direct repeats within the CRE. In contrast, F9 extracts exhibited factor binding to only two of the three sequences and lack a major factor detected in the above two cell types. Protein-binding sites within each of the three sequences were identified by methylation interference experiments. These data were in full agreement with results obtained by a competition assay performed with a series of mutant oligonucleotides containing a few nucleotide substitutions in each of the three regions. The results illustrate complex DNA-protein interactions in which several independent proteins bind to overlapping sequences in the CRE in a cell type-specific fashion.

Animals↗
PubMed1995

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.

Base Sequence↗
PubMed1989

Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.

Base Sequence↗
PubMed2001

Alteration of a sequence with homology to human endogenous retrovirus (HERV-K) in primary human glioma: implications for viral repeat mediated rearrangement.

We had earlier demonstrated that a comparison of DNA fingerprinting profiles of tumor and corresponding normal DNA from the same patient by random amplified polymorphic DNA (RAPD) analysis can readily demonstrate alterations in tumor DNA [Gene 206 (1998) 45 and J. Neuro Oncol. 48 (2000) 1]. These alterations could be used to identify changes in tumor DNA where the prior identity of the locus was not known. In this study, we report the identification, cloning and characterization of a RAPD amplified fragment which was lost in a glioma, a grade IV glioblastoma multiforme (GBM). Comparison of the RAPD profile of tumor and corresponding leucocyte DNA revealed several differences between the two. These included a band of 443 bases, which was demonstrated in the normal, but not in tumor DNA. On sequencing, this band was found to be homologous with a group of SINE sequences, which are probably derived from the human endogenous retrovirus-K (HERV-K). Homology search also reveals that HERV-K-derived sequences are interspersed, amongst others, in the tumor suppressor gene BRCA2 and the DNA repair gene XRCC1. Of particular interest is the inverted repeat pattern of HERV-derived sequences in the genes. While not demonstrating a cause effect relationship, this highlights the possible role of such virus-derived sequences in gene inactivation by recombination during tumorigenesis.

Base Sequence↗
PubMed1998

Identification of two linear plasmids in the actinomycete Planobispora rosea.

Two linear plasmids (pPR1, 27.5 kb, and pPR2, 16 kb) were identified in Planobispora rosea, an actinomycete that produces the antibiotic GE2270, an inhibitor of the elongation factor Tu. Strains lacking both plasmids still produce and are resistant to GE2270. The two plasmids share an internal region of high similarity, but no cross-hybridization was detected between their telomeric regions or between plasmid and chromosomal DNA. The 5' ends of the plasmids appear to be linked to terminal proteins. The telomeric regions of pPR2 were cloned after 3'-end homopolymer tailing and PCR amplification. The approximately 650 nt telomeric DNA sequences of pPR2 are repeated in inverted orientation and are rich in direct and inverted repeats; the 350 bp terminal region is less G + C-rich than the rest of the plasmid. The structural organization of these plasmids appears to be similar to Streptomyces linear replicons.

Actinomycetales↗
PubMed1999

Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2 deduced from mRNA nucleotide sequences.

Tol2 is a terminal-inverted repeat transposable element of the medaka fish Oryzias latipes. It is one of a few elements of this class so far demonstrated to be active in vertebrates, thus providing a unique tool for establishing a gene tagging system. For the purpose of identifying its transposase, we analyzed the structures of mRNAs originating from the Tol2 element. The results indicated that transcription of Tol2 is initiated at several sites, the four open reading frames in Tol2 roughly corresponding to exons, and that two main forms of mRNAs, covering exons 1-4 and exons 2-4, are present in medaka fish cells. One or both of these mRNAs are likely to encode a transposase, the amino acid sequence of which was deduced.

Amino Acid Sequence↗
PubMed2006

Unusually long palindromes are abundant in mitochondrial control regions of insects and nematodes.

BACKGROUND: Palindromes are known to be involved in a variety of biological processes. In the present investigation we carried out a comprehensive analysis of palindromes in the mitochondrial control regions (CRs) of several animal groups to study their frequency, distribution and architecture to gain insights into the origin of replication of mtDNA. METHODOLOGY/PRINCIPAL FINDINGS: Many species of Arthropoda, Nematoda, Mollusca and Annelida harbor palindromes and inverted repeats (IRs) in their CRs. Lower animals like cnidarians and higher animal groups like chordates are almost devoid of palindromes and IRs. The study revealed that palindrome occurrence is positively correlated with the AT content of CRs, and that IRs are likely to give rise to longer palindromes. CONCLUSIONS/SIGNIFICANCE: The present study attempts to explain possible reasons and gives in silico evidence for absence of palindromes and IRs from CR of vertebrate mtDNA and acquisition and retention of the same in insects. Study of CRs of different animal phyla uncovered unique architecture of this locus, be it high abundance of long palindromes and IRs in CRs of Insecta and Nematoda, or short IRs of 10-20 nucleotides with a spacer region of 12-14 bases in subphylum Chelicerata, or nearly complete of absence of any long palindromes and IRs in Vertebrata, Cnidaria and Echinodermata.

AT Rich Sequence↗
PubMed2003

Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

Animals↗