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PubMed1979

Subunit structure of alpha-satellite DNA containing chromatin from African green monkey cells.

alpha-Satellite DNA containing chromatin from African green monkey cells (CV-1 cells) has been used to study the question whether or not nucleosomes are arranged in phase with the 172 bp repeat unit of the satellite DNA. Digestion experiments with DNAase II led us to exclude a simple phase relationship between the nucleosomal and the satellite DNA repeats. Digestion of CV-1 nuclei with micrococcal nuclease and endogenous nuclease (s) produced a series of sharp bands in the satellite DNA register over a background of heterogeneous length fragments. This observation is explained by a preferential cleavage of certain nucleotide sequences by these nucleases and is not in contradiction to our conclusion that a simple phase relationship does not exist.

Animals↗

PubMed1999

Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions.

Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.

DNA Replication↗

PubMed1995

Pseudomonas aeruginosa outer membrane protein OprF as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope.

OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been proposed to be comprised of a series of beta-strands separated by periplasmic or surface-exposed loop regions. In this study, a simple malarial epitope was used to demonstrate that OprF can be used as an expression vector to present foreign peptide sequences, namely, the 4-amino-acid (aa) repeating epitope (Asn-Ala-Asn-Pro = NANP) of the circumsporozoite protein of the human malarial parasite Plasmodium falciparum. Eight permissive sites, that allowed the expression and surface exposure of the malarial epitope, were identified throughout OprF. Using a monoclonal antibody (mAb) specific for the malarial epitope, we investigated the effects of positioning and length of the epitope on its antigenicity in the OprF expression vector system. It was demonstrated that the malarial epitope inserted at aa26 was significantly more reactive with the epitope-specific mAb (i.e., more antigenic) when assayed in the context of whole cells whereas those at aa213 and aa290 were more antigenic when assayed in the OM. The malarial epitope inserted at aa188 and aa196 was moderately antigenic, while this epitope inserted at aa215 and aa310 showed low antigenicity with the same mAb in both whole cell and OM assays. For two insertion sites, aa26 and aa213, we demonstrated that the insertion of multiple copies of the epitope enhanced reactivity with the malarial epitope-specific mAb. These data are discussed with respect to the local OprF sequences into which the epitope was inserted.

Amino Acid Sequence↗

PubMed1994

In vitro identification of a protein of Saccharomyces cerevisiae that interacts specifically with the G-rich DNA strand of the telomere.

The telomeres of Saccharomyces cerevisiae consist of a repeated G2-3T(GT)1-6 DNA sequence that forms a complex with proteins. To date only the RAP1 protein has been shown to bind to the simple sequences in yeast telomeric DNA, as well as to non-telomeric regulatory sites. We have used synthetic oligodeoxyribonucleotides, both double- and single-stranded, to identify specific yeast telomeric proteins in a partially purified yeast extract. Using the gel shift assay, we detected a binding activity that is stable at high ionic strength and that recognizes specifically the G-rich protrusion of a double-stranded synthetic yeast telomere, as well as the G-rich single strand. This is the first evidence of a purely telomeric protein in that it binds to the single-stranded telomeric protrusion of the yeast chromosome.

DNA, Fungal↗

PubMed2005

Association of ANKH gene polymorphisms with radiographic hand bone size and geometry in a Chuvasha population.

We performed a family-based association study to test the hypothesis that genetic variation at the human orthologue of the mouse progressive ankylosis gene (ANKH) is involved in determining bone size (BS) and bone geometry (BG). The study population comprised 126 nuclear families with 574 adult Chuvashian individuals living in small villages in the Russian Federation. Quantitative bone traits were determined by analyzing plain hand radiographs. Familial correlations for all studied traits revealed a high degree of heritability in this ethnically homogeneous population. Three simple tandem repeat (STR) polymorphisms, one intragenic and two flanking markers, as well as six single nucleotide polymorphisms (SNPs) were tested. The SNPs were detected by re-sequencing experiments and covered ANKH exons with their flanking splice sites and the promoter region. We used three different transmission disequilibrium tests (TDTs) and obtained multiple significant association signals for all investigated bone traits. Alleles of several markers located at different positions of the ANKH locus, including the promoter, consistently revealed the association. The bone traits tested are closely related to bone fragility suggesting a role for ANKH in osteoporosis.

Adolescent↗

PubMed2026

Spacer-engineered donor DNA enhances CRISPR-Cas9-mediated knockin to establish a chemical knockdown platform for endogenous proteins.

Precise installation of functional protein domains at endogenous loci is a powerful approach for interrogating protein functions, but its broad application is limited by the low efficiency of homology-directed repair (HDR)-mediated knockin during Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing. Here, we investigated a simple donor DNA engineering strategy that enhances HDR-mediated gene knockin by appending additional gRNA-recognizable spacer sequences to donor templates. Systematic analysis of linear dsDNA and plasmid donors showed that spacer position, length, and orientation influenced HDR efficiency, and that spacer-containing donors improved knockin across multiple genomic loci, insertion sizes, cell types and delivery modalities. Mechanistic analyses revealed that spacer-containing donors formed stable complexes with Cas9/gRNA and showed increased nuclear localization, supporting nuclear delivery as a key contributor to improved editing outcomes. We then applied this gene-editing strategy to establish a chemical knockdown platform by installing drug-responsive degrons at endogenous loci, generating cell lines in which GSK3β or Lin28A protein could be rapidly, potently and reversibly depleted by drug treatment. These platforms enable selective modulation of endogenous proteins and reveal cellular responses that may differ from those obtained using conventional genetic perturbation. Together, this work establishes a readily implementable framework that integrates improved gene editing with on-demand chemical knockdown of endogenous proteins.

CRISPR-Cas9↗

PubMed2006

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece.

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.

Adolescent↗

PubMed1993

Characterization of two chromosome 12 cosmid libraries and development of STSs from cosmids mapped by FISH.

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LL12NC01 and LL12NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.

Base Sequence↗

PubMed1997

Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction.

Clonal rearrangements of the Ig heavy chain (IGH) locus consisting of either intrachromosomal (VDJ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14;18)(q32;q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3' BCL2 breakpoint cluster region. The final case fell immediately 3' of the 3' UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11;14)(q13;q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpointsfell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.

Base Sequence↗

PubMed2002

Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR.

OBJECTIVE: Quantitative fluorescence-polymerase chain reaction (QF-PCR) has recently been used for the detection of common chromosomal aneuploidies in prenatal diagnosis. Here we describe our experience in prenatal diagnosis of 1100 samples. METHODS: Extraction of DNA was performed from amniotic fluid, chorionic villus samples (CVS), fetal blood and fetal tissue samples, using a simple, rapid protocol. Fluorescent multiplex PCR products of single tandem repeats (STRs) located on chromosomes 13, 18, 21, X and Y were then analyzed on an automated laser fluorescent sequencer. All samples were analyzed with at least two polymorphic markers for chromosomes 13, 18 and 21 and one for the X chromosome. The amelogenin locus was used for sexing. Analysis was performed twice on affected samples. When miscellaneous results were obtained extra markers were used. RESULTS: We evaluated the usefulness of different markers in the Greek population. In a total of 1100 samples, 25 chromosome aberrations were identified, including trisomy 13, 18 and 21, XYY, triploidies 69,XXX and 69,XXY and one Turner mosaic. All results but three were consistent with conventional cytogenetic analysis. One mosaic was missed. Most bloodstained samples were successfully analyzed. CONCLUSION: Successful analysis of a large number of prenatal samples proves QF-PCR to be an efficient adjunct in routine prenatal diagnosis.

Adult↗

PubMed1996

Single-cell analysis of unstable genes.

PURPOSE: We have developed sensitive diagnostic procedures for studies on the normal and mutant alleles of the triplet repeat genes associated with myotonic dystrophy and fragile X in single human somatic cells, gametes and embryos. METHODS: Polymerase chain reaction (PCR) assays for the normal alleles of the myotonic dystrophy and fragile X loci have been refined to the sensitivity of the single cell. In addition, we have developed a simple PCR-based technique, termed ¿Repeat Primer PCR', which can detect the full fragile X expansion in small samples of buccal cells. CONCLUSIONS: The assay for the triplet repeat sequence in the myotonic dystrophy locus could not be used to study stability since we observed additional PCR products derived from in vitro expansion of the triplet repeat sequence during the PCR reaction itself. The implications of in vitro expansion and allele drop-out for studies on the timing of the expansion in development and preimplantation diagnosis of triplet repeat diseases are discussed. The development of a new PCR procedure to identify the expanded alleles of the fragile X locus could prove invaluable for monitoring the timing of repeat expansion in early embryonic development. Triplet repeat polymorphisms provide a means of identifying the maternally and paternally-derived alleles of the myotonic dystrophy gene. Using single cell reverse transcriptase PCR analysis, we have monitored the onset of the myotonic dystrophy gene transcription in early preimplantation embryos. Transcripts from the paternally-inherited allele of the myotonic dystrophy gene are already detectable in the 1-cell stage human embryo.

Alleles↗

PubMed2002

Electric brain response to sound repetition in humans: an index of long-term-memory - trace formation?

The developing auditory system is exposed to an almost endless variation of acoustic input in its every-day environment. All these different stimuli cannot, of course, be encoded in memory, however, at least not for any longer time. The present data suggest a principle that might be followed by the brain in selecting stimuli for the formation of longer-duration memory traces. Normal subjects ignoring auditory stimulation were presented with a randomized sequence of different sounds (simple tones in Experiment I and frequency glides in Experiment 2) with the exception that every now and then, randomly, one of the sounds was repeated in a row a few times. It was found that these repetitions elicited a novel electric brain response ('repetition negativity') which might be generated by neural activity forming these longer-duration traces. This would suggest that of all the different sounds, only those that soon occur again form more enduring memory traces (when sounds have no particular significance).

Acoustic Stimulation↗

PubMed1983

The independent evolution of two closely related satellite DNA elements in rats (Rattus).

We have identified and determined the sequence and organization of a new rat satellite DNA in Rattus rattus, the roof rat. This satellite DNA, which we call R. rattus satellite I', consists of tandem arrays of a 185 base pair (bp) repeat unit that we call a'. a' is 86% homologous to a 185 bp portion of the 370 bp repeat unit of the previously described rat satellite [Pech et al. (1979) Nucleic Acids Res. 7, 417-432] present in the common laboratory rat species, R. norvegicus. We can thereby distinguish two 185 bp portions of the satellite I 370 bp repeat unit: "a" (homologous to a') and "b". Satellite I has the structure (a,b)n, and satellite I' has the structure (a')n. Like a, a' is only about 60% homologous to b and fails to hybridize to b. Although R. norvegicus and R. rattus contain about the same total concentration of satellite sequences, R. norvegicus contains essentially only the a,b type (satellite I), whereas R. rattus contains a' type (satellite I') and lesser amounts of the a,b type (satellite I). The a,b type (satellite I) in R. rattus is very similar to that in R. norvegicus as judged both by hybridization and by the presence of all but one of the major restriction enzyme sites that characterize the R. norvegicus satellite I. In R. rattus the a' and a,b repeat units are not detectably present in the same tandem array. All of the sequence differences between a' (R. rattus) and a (R. norvegicus) can be accounted for by simple base substitutions, and the implication of this and other features of rat satellite DNA structure for satellite DNA evolution are discussed.

Animals↗

PubMed2002

A chromosomal region 7p11.2 transcript map: its development and application to the study of EGFR amplicons in glioblastoma.

Cumulative information available about the organization of amplified chromosomal regions in human tumors suggests that the amplification repeat units, or amplicons, can be of a simple or complex nature. For the former, amplified regions generally retain their native chromosomal configuration and involve a single amplification target sequence. For complex amplicons, amplified DNAs usually undergo substantial reorganization relative to the normal chromosomal regions from which they evolve, and the regions subject to amplification may contain multiple target sequences. Previous efforts to characterize the 7p11.2 epidermal growth factor receptor ) amplicon in glioblastoma have relied primarily on the use of markers positioned by linkage analysis and/or radiation hybrid mapping, both of which are known to have the potential for being inaccurate when attempting to order loci over relatively short (<1 Mb) chromosomal regions. Due to the limited resolution of genetic maps that have been established through the use of these approaches, we have constructed a 2-Mb bacterial and P1-derived artificial chromosome (BAC-PAC) contig for the EGFR region and have applied markers positioned on its associated physical map to the analysis of 7p11.2 amplifications in a series of glioblastomas. Our data indicate that EGFR is the sole amplification target within the mapped region, although there are several additional 7p11.2 genes that can be coamplified and overexpressed with EGFR. Furthermore, these results are consistent with EGFR amplicons retaining the same organization as the native chromosome 7p11.2 region from which they are derived.

Blotting, Northern↗

PubMed1991

Circularization of human immunodeficiency virus type 1 DNA in vitro.

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.

Bacteriophage phi X 174↗

PubMed1991

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Animals↗

PubMed1998

Variants of the 3' region of the cagA gene in Helicobacter pylori isolates from patients with different H. pylori-associated diseases.

The CagA protein of Helicobacter pylori is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3' region of the cagA gene contains repeated sequences. To determine whether there are structural changes in the 3' region of cagA that predict outcome of H. pylori infection, we examined 155 cagA gene-positive H. pylori isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3' region of the cagA gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3' region of the cagA gene in Japan differs markedly from the primary sequence of cagA genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (P < 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in cagA genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among cagA-positive H. pylori strains.

Adult↗

PubMed1984

Genetic recombination of Xenopus laevis 5 S DNA in bacteria.

The behavior in genetic recombination of Xenopus laevis 5 S DNA has been examined, with particular emphasis on the role of 15-base-pair tandem repeats in the A + T-rich spacer. Fragments of 5 S DNA were introduced into Escherichia coli cells as inserts in the recombination vectors, lambda rva and lambda rvb. Intermolecular recombinants were selected in which, because of properties of the phage vectors, the crossover event must have occurred within the 5 S DNA inserts. Inserts from individual recombinants have been characterized in detail. The effects of varying the number (n) of 15-base-pair repeats and the recombination capabilities of the phage and host have been investigated. In these crosses, unequal crossovers can occur, yielding inserts different in size from the parental inserts. When the number of 15-mers is large (n = 12 or 20), most of the unequal crossovers have occurred within the 15-mers, resulting in an altered n value, although other homologies within the 5 S DNA sequence can also support unequal events. Increasing n in the parental inserts modestly increases the overall frequency of recombination and the percentage of altered inserts. We conclude that, in a bacterial setting, the 15-base-pair repeats stimulate recombination only slightly by allowing alternative registers for heteroduplex formation. The degree of stimulation observed is less than predicted by one simple model.

Animals↗

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