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PubMed2001

Genome quality control: RIP (repeat-induced point mutation) comes to Podospora.

RIP (repeat-induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C-G to T-A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP-like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating-type region. The 9 kb sequenced show 106 C-G to T-A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP-like event extended to single-copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C-methylation. Interestingly, the N. crassa zeta-eta sequence, which acts as a potent de novo C-methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.

Ascomycota↗
PubMed1994

Purification of recombinant Shiga-like toxin type I A1 fragment from Escherichia coli.

Shiga-like toxin I A1 (Slt-IA1) is a RNA N-glycosidase which depurinates a specific adenosine of 28 S eukaryotic rRNA thus inhibiting protein synthesis and ultimately leading to cell death. We have overexpressed this protein in Escherichia coli using a high copy number plasmid and purified the enzyme to homogeneity using a three-step process. Slt-IA1 is released from the periplasm of cells using polymyxin B sulfate, precipitated with ammonium sulfate, and adsorbed to a Matrex Gel Green A dye-ligand agarose column. The enzyme is eluted from the Green A agarose as a single peak with 0.32 M NaCl. Slt-IA1 was purified approximately 1979-fold and routinely gave yields greater than 100%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular weight of 28,000. An isoelectric point of 5.1 was determined using analytical isoelectric focusing gels. In an in vitro protein synthesis inhibition assay, 0.02 pM of purified Slt-IA1 inhibited protein synthesis by 50%.

Animals↗
PubMed1994

The structure of the rDNA intergenic spacer of Avena sativa L.: a comparative study.

The sequence of the 18S-25S ribosomal RNA gene intergenic spacer (IGS) of Avena sativa was determined. DNA was cloned after polymerase chain reaction amplification of the IGS. The spacer of 3980 bp is composed of non-repeated sequences and five tandem arrays of repeated sequences, named A to E. Homology between oats IGS and other grass species was found. Tandem arrays D and E seem to be originated by duplication from single-copy sequences in related species. A transcription initiation site and putative sites of termination, processing and methylation were detected by computer-aided search. These sites resemble motifs conserved in the IGS of plant species.

Base Sequence↗
PubMed1993

Characterization of an aspartic proteinase of Mucor pusillus expressed in Aspergillus oryzae.

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1-2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-beta-N-acetylglucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.

Aspartic Acid Endopeptidases↗
PubMed2003

Relative rates of nucleotide substitution in frogs.

Accurate estimation of relative mutation rates of mitochondrial DNA (mtDNA) and single-copy nuclear DNA (scnDNA) within lineages contributes to a general understanding of molecular evolutionary processes and facilitates making demographic inferences from population genetic data. The rate of divergence at synonymous sites ( K(s)) may be used as a surrogate for mutation rate. Such data are available for few organisms and no amphibians. Relative to mammals and birds, amphibian mtDNA is thought to evolve slowly, and the K(s) ratio of mtDNA to scnDNA would be expected to be low as well. Relative K(s) was estimated from a mitochondrial gene, ND2, and a nuclear gene, c-myc, using both "approximate" and likelihood methods. Three lineages of congeneric frogs were studied and this ratio was found to be approximately 16, the highest of previously reported ratios. No evidence of a low K(s) in the nuclear gene was found: c-myc codon usage was not biased, the K(s) was double the intron divergence rate, and the absolute K(s) was similar to estimates obtained here for other genes from other frog species. A high K(s) in mitochondrial vs. nuclear genes was unexpected in light of previous reports of a slow rate of mtDNA evolution in amphibians. These results highlight the need for further investigation of the effects of life history on mutation rates.

Animals↗
PubMed1981

The tyrT locus: termination and processing of a complex transcript.

The tyrT locus of E. coli contains a 208 bp spacer region that separates two copies of sequence encoding tRNATyr1. The spacer includes a 120 bp sequence that is homologous to a sequence that is repeated three times in the distal portion of the tyrT locus. The tyrT locus possesses a graded set of transcription termination sites that are spaced at 180 base intervals, corresponding to the distal repeated gene structure. The major termination site occurs within the second repeat unit, 225 bases beyond the mature tRNA sequences. In the presence of a temperature-sensitive rho protein there is increased read-through at this site to a termination site located 180 bases downstream in the third repeat and to several termination sites even further downstream. The primary native transcript, in the region distal to the second tRNA, carries the information for a low molecular weight, extremely basic protein. Although analogous coding sequences are present in the spacer and other repeat units, because of single base substitutions these sequences are pseudogenes. The parallel between the tyrT and TyrU gene clusters is discussed in relation to dual function transcripts that specify both tRNA and protein.

Bacterial Proteins↗
PubMed2001

Differential gating of slow postsynaptic and high-frequency spike-like components in human somatosensory evoked potentials under isometric motor interference.

Human cortical somatosensory evoked potentials (SEP) can be modified by concomitant motor tasks ('gating'), through peripheral occlusion and/or central mechanisms. The present study aimed (1) at refining earlier results concerning motor-gating of the primary cortical EPSP-related N20 response after electric median nerve stimulation, and (2) at providing first data on motor-gating of the 600 Hz SEP wavelet burst which occurs superimposed onto N20 and primarily reflects repetitive cerebral population spikes. In 12 healthy subjects median nerve SEP were elicited, using electrical stimuli with intensities below, at and above motor threshold, under either rest or an isometric fist clenching task. Amplitude and latency modifications were analysed for the peripheral compound action potential (CAP), low-frequency SEP components (N20, P25, N35 and P70) and the high-frequency burst. While the peripheral CAP remained unchanged, isometric motor innervation significantly attenuated N20, P25 and P70 amplitudes and shortened peak latencies progressively for all components after N20. In contrast, the high-frequency 600 Hz burst was modulated neither in amplitude nor in latency. Regular amplitude recruitment occurred for all components independent from the motor task, excluding channel saturation as an explanation for gating. We suggest that SEP gating under isometric motor innervation is a central process which selectively operates on specific SEP components and could partly reflect an "efference copy" mechanism.

Action Potentials↗
PubMed2004

The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats.

Metastasis of diseased cells is the basic event leading to death in individuals with cancer. Establishment of metastasis requires that tumour cells migrate from the site of the primary tumour into the circulation system, escape from the vasculature and form secondary tumours at novel sites. These processes depend to a large degree on cytoskeletal remodeling. We show here that multiple copies of the short actin-binding module CLIK(23) from human or Caenorhabditis elegans calponin proteins effectively inhibit cell motility on two dimensional matrices and suppress soft agar colony formation of metastatic melanoma and adenocarcinoma cells of murine and human origin. Ectopic expression of CLIK(23) modules for 30 days results in the formation of multinucleated cells. The repeat displays true modular behaviour, resulting in increased cytoskeletal effects in direct correlation with the increase in number of modules. Our results demonstrate that the role of calponin in the signature profile of metastasising cells is that of a mechanical stabiliser of the actin cytoskeleton, which interferes with actin turnover by binding at a unique interface along the actin filament.

Adenocarcinoma↗
PubMed1997

A multicenter longitudinal clinical trial of a new system for restorations.

STATEMENT OF PROBLEM: A new method for fabrication of crowns and fixed partial dentures (Procera system) that involves electric discharge machining and copy milling has been developed. The metal used is unalloyed titanium, which can be processed as a single coping or multiple units joined to a pontic by laser welding. PURPOSE: The single-unit coping or the fixed partial denture (FPD) substructure is then veneered with a low-fusing porcelain. MATERIAL AND METHODS: In this article the clinical application of this technique was evaluated by six major universities in the United States. A total of 114 patients participated in this study, which involved 126 restorations (55 maxillary and 71 mandibular prostheses). There were 179 abutments, of which 73 were crowns and 53 were three-unit FPDs. Surface and color, anatomic form, and margin integrity were assessed 1 month after cementation and at 1 year with the California Dental Association (CDA) quality assessment evaluation system. RESULTS: No statistically significant differences in CDA scores between the 1 month evaluations and the 1 year assessments were found for surface and color (p = 0.68), anatomic form (p > 0.99), or margin integrity (p = 0.57). By use of the lowest ranking in the three categories as the overall quality of the restoration, only 3.3% (two crowns and two FPDs) were not acceptable at the 1-month visit and 4.5% (two crowns and three FPDs) were not acceptable at the 1-year evaluation. At-the 1-month visit 96.6% (114) of the restorations were considered to be satisfactory, whereas 95.5% (107 restorations) were evaluated similarly at the 1-year evaluation. CONCLUSIONS: The Procera system demonstrated, by use of the CDA criteria, its capability to produce quality prostheses that were rated satisfactory more than 95% of the time after insertion and maintained this high rating at least for 1 year.

Adolescent↗
PubMedDate not supplied

Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age.

The chronological accumulation of mitochondrial dysfunction has been proposed as a potential mechanism in the physiological processes of aging. Cytochrome c oxidase deficient, succinate dehydrogenase positive muscle fibers containing high copy numbers of a mitochondrial DNA mutation are a pathological hallmark of mitochondrial DNA disorders. We show that there is an age-related increase in cytochrome c oxidase-deficient cells in both hippocampal pyramidal neurons and choroid plexus epithelial cells. We suggest that these cells contribute to the cell death and dysfunction in CNS aging.

Adolescent↗
PubMed1994

Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR.

To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin-embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT-PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin-embedded liver biopsies.

Base Sequence↗
PubMed1998

Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea.

cDNAs encoding chitinases were cloned and characterized from Bombyx mori and Hyphantria cunea, and their gene expression during the metamorphosis was also studied. The chitinase cDNA from B. mori encodes a protein of 565 amino acids with a calculated molecular mass of 63.4 kDa and the H. cunea chitinase cDNA encodes a protein of 553 amino acids with a calculated molecular mass of 62.0 kDa. Amino acid alignment of the two chitinases revealed 75% homology and 77-80% with M. sexta chitinase. The putative cleavage site of the signal peptide was between amino acid residues 20 and 21 for both chitinases. There were three potential N-glycosylation sites in the chitinase of B. mori at the amino acid residues 86-89, NFTS 304-307, NATG, 398-401, NYTV, whereas two potential N-glycosylation sites were present at the amino acid residues 86-89, NFTA and 304-307, NATG, in that of H. cunea. Southern blot analysis of total genomic DNA suggested that the B. mori genome has only one chitinase gene detectable by the cDNA probe and the H. cunea genome has one or two chitinase gene copies. Northern analysis indicated that gene expression was up-regulated during the molting process, larval-pupal transformation and pupal-adult transformation, when enzymatic degradation of cuticle was occurring.

Amino Acid Sequence↗
PubMed1998

Structure and expression of several bean (Phaseolus vulgaris) nuclear transfer RNA genes: relevance to the process of tRNA import into plant mitochondria.

Bean nuclear genes for tRNA(Pro), tRNA(Thr) and tRNA(Leu) were isolated. Expression of the tRNA(Pro) genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNA(Pro) gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNA(Thr) genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNA(Thr). The tRNA(Leu) gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNA(Leu) however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNA(Leu) and tRNA(Thr). From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.

Base Sequence↗
PubMed1988

Spliceosomal RNA U6 is remarkably conserved from yeast to mammals.

The small nuclear RNA U6 and its gene have been isolated from yeast. In striking contrast to other yeast spliceosomal RNAs, U6 is very similar in size, sequence and structure to its mammalian homologue. The single-copy gene is essential. These properties suggest a central role in pre-mRNA processing. An extensive base-pairing interaction with U4 snRNA is described; the destabilization of the U4/U6 complex seen during splicing thus requires a large conformational change.

Base Sequence↗
PubMed2005

Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability.

Aurora kinases are key regulators of chromosome segregation during mitosis. We have previously shown by microarray analysis of primary lung carcinomas and matched normal tissue that AURKB (22 out of 37) and AURKA (15 out of 37) transcripts are frequently over-represented in these tumours. We now confirm these observations in a second series of 44 carcinomas and also show that aurora B kinase protein levels are raised in the tumours compared to normal tissue. Elevated levels of expression in tumours are not a consequence of high-level amplification of the AURKB gene. Using a coding sequence polymorphism we show that in most cases (seven out of nine) tumour expression is predominantly driven from one AURKB allele. Given the function of aurora B kinase, we examined whether there was an association between expression levels and genetic instability. We defined two groups of high and low AURKB expression. Using a panel of 10 microsatellite markers, we found that the group showing the higher level of expression had a higher frequency of allelic imbalance (P=0.0012). Analysis of a number of other genes that are strongly and specifically expressed in tumour over normal lung, including SERPINB5, TERT and PRAME, showed marked allelic expression imbalances in the tumour tissue in the context of balanced or only marginally imbalanced relative allelic copy numbers. Our data support a model of early carcinogenesis wherein defects in the process of inactivation of lung stem-cell associated genes during differentiation, contributes to the development of carcinogenesis.

Allelic Imbalance↗
PubMed2003

Apparent intrachromosomal exchange on the human Y chromosome explained by population history.

The human Y chromosome displays an unusual content of repetitive sequences. Y-chromosomal repeats are potential targets for intrachromosomal recombination, which is thought to be involved in a number of Y-associated defects, such as male infertility. Such rearrangements could potentially be investigated by the use of highly polymorphic DNA markers located within the repeat units, such as microsatellites. Here we analyse the two copies of the Y-chromosomal microsatellite DYS385, which we identified and localized to an approximately 190 kb duplicated and inverted fragment at Yq11.223. We found a highly significant correlation (r=0.853, P&<0.001) and a nonsignificant difference in a chi(2)-test (chi(2)=15.45, P>0.05) between the allele frequency distributions at both copies of the Y-STR in a German population sample (n=70). Such nearly identical allele frequency distribution between two copies of a duplicated highly polymorphic microsatellite cannot be explained by the independent mutational process that creates microsatellite alleles. Instead, this might be interpreted as evidence for a reciprocal intrachromosomal exchange process between the duplicated fragments. However, more detailed analyses using additional human populations as well as additional Y chromosome markers revealed that this phenomenon is highly population-specific and disappears completely when Y-STR diversity is analysed in association with two Y-SNP haplogroups. We found that the diversity of the two DYS385 loci (and other Y-STRs) is highly depending on the haplogroup background, and that equal proportions of both haplogroups in the German sample explains the nearly identical allele frequency distributions at the two DYS385 loci. Thus, we demonstrate here that allele frequency distributions at duplicate loci that are suggestive of intrachromosomal recombination can be explained solely by population history.

Chromosome Mapping↗
PubMed2002

Engineered herpes simplex virus 1 is dependent on IL13Ralpha 2 receptor for cell entry and independent of glycoprotein D receptor interaction.

In the first stage of engineering a herpes simplex virus (HSV)-1 that specifically targets human malignant glioma cells, we constructed a recombinant virus designated R5111 in which we have ablated the binding sites for sulfated proteoglycans in glycoproteins B and C, replaced the amino-terminal 148 aa in glycoprotein C by IL-13 flanked at its amino terminus with a signal peptide, and inserted a second copy of IL-13 after the amino acid 24 of glycoprotein D. In the process, the binding site for HveA, a viral entry receptor, was disrupted. We have also transformed a cell line (J1.1) lacking HSV-1 receptors to express IL13Ralpha2 receptor (J13R cells). We report the following: the R5111 recombinant virus replicates as well as wild-type virus in a variety of cell lines including cell lines derived from brain tumors. R5111 failed to replicate in the parent J1.1 cell line but multiplied to titers similar to those obtained in other cell lines in the J13R cell line. On the basis of the evidence that R5111 can use the IL13Ralpha2 receptor for entry, we conclude that HSV-1 can use receptors other than HveA or nectins, provided it can bind to them. The domains of gD that interact with HveA and nectin receptors are independent of each other. Lastly, the fusogenic activities of the glycoproteins in the viral envelope are not dependent on a set of unique interactions between glycoprotein D and its receptor. The construction of R5111 opens the way for construction of viruses totally dependent on selected receptors for entry or imaging of targeted cells.

Base Sequence↗
PubMed1991

De novo chromosome formation in rodent cells.

A hybrid cell line was produced by the fusion of an EC3/7 mouse cell with a Chinese hamster ovary cell. The EC3/7 cell carries a dicentric chromosome with a functional marker centromere. This marker centromere contains human, lambda, and bacterial vector DNA sequences and a dominant selectable gene (aminoglycoside 3'-phosphotransferase type II; neo). In the hybrid, the marker centromere separated from the dicentric chromosome and formed a full-sized chromosome (lambda neo). The newly formed chromosome is stable, even under nonselective culture conditions. This functional chromosome, which is the result of an amplification process, is composed of seven large, different-sized amplicons. Each amplicon contains multiple copies of human, lambda, neo, and mouse telomeric DNA sequences. Individual amplicons are separated from each other by mouse major satellite DNA sequences. The marker centromere was localized to a terminal amplicon by anticentromere immunostaining. The number of amplicons in the newly formed chromosome is remarkably consistent. This finding suggests that the length of the newly formed chromosome is highly constrained.

Animals↗