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PubMed2000

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae.

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8(m)) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

DNA Mutational Analysis↗

PubMed2001

A novel method to isolate the common fraction of two DNA samples: hybrid specific amplification (HSA).

Hybrid specific amplification (HSA) is a novel simple method elaborated in order to isolate the common fraction of two DNA samples while avoiding the background due to repeated sequences. The method is based on the suppressive PCR principle, associated with a Cot1 pre-hybridization step. In recent work we demonstrated that hyperprolificity observed in Booroola ewes is associated with a mutation in the bone morphogenetic protein receptor IB gene (BMPR-IB). We applied HSA between ovarian cDNA and DNA from four BAC clones containing BMPR-IB in order to test for the presence of other genes expressed in ovary and to isolate additional BMPR-IB exon sequences. Of the 460 clones obtained, none contained repeated sequences. We successfully obtained 37 clones representing the major part of BMPR-IB coding sequence, together with 5'- and 3'-UTR sequences. Here we have successfully applied HSA to a particular tissue, but it should be possible to trap the common fraction of two DNA samples, whatever their nature.

Animals↗

PubMed1994

Head waving in Aplysia californica. I. Behavioural characterization of searching movements.

Searching is an important component of several types of goal-directed behaviour. In soft-bodied animals, searching behaviour can appear quite complex because the range of body movement is not constrained by joints, limbs or muscles with discrete areas of origin and insertion. In addition, animals exhibiting this type of behaviour utilize their maximum freedom of movement. In this paper, we describe the head-waving searching behaviour of the sea hare Aplysia californica by characterizing patterns of movements and the changes in body shape that underlie these movements. A bout of head waving consists of a series of discrete movements separated by pauses. Each discrete movement lasts 4-10s and is directed either horizontally or vertically with respect to stationary part of the animal. Large movements, such as bending from the extreme right to the extreme left, consist of a series of these shorter movements separated by pauses lasting 1-2s. In all head-waving movements, the transverse axis of the head is kept relatively parallel with the substratum. Thus, vertical movements require only bending of the body, whereas horizontal movements require twisting of the body, particularly when the posture is more erect. During head waving, the anterior two-thirds of the body is free to move, but most of the bending occurs immediately posterior to the head region. There is no periodicity of movements within a bout of head waving, although isolated instances of repeated movements are sometimes observed. Therefore, although the individual movements during head waving are relatively simple, the absence of a patterned sequence accounts for the complexity of the overall behaviour. These observations both constrain models of the neural organization of head waving and provide criteria for categorizing head-waving movements in further behavioural and physiological studies.

Animals↗

PubMed2005

[Genetic diagnosis of Huntington disease].

We report here a simple and effective method to assess the CAG repeat size of HD gene for gene diagnosis of Huntington disease. Genomic DNA sequences in polymorphic CAG repeat HD gene was amplified by PCR with TaKaRa LA Taq DNA polymerase and GC buffer. To distinguish normal alleles from HD alleles, DNA fragments of affected alleles were recovered as templates for secondary PCR. The secondary PCR products were cloned into T vector for sequencing to determine CAG repeat size. A total of 20 normal individuals and 3 members from a HD pedigree were included in this study. Results showed that the CAG repeat numbers in 20 normal individuals and 3 normal alleles from the HD pedigree varied in normal range, while in 3 HD alleles, the copy numbers of CAG repeat were 39, 40, 41 respectively. There was no overlap between the copy number of the normal and affected alleles. In conclusion, the TaKaRa LA Taq DNA polymerase with GC buffer can be used to effectively amplified CAG repeat of HD gene, which combined DNA sequencing can diagnose HD accurately. In addition, these finding suggested that the dynamic mutation in HD gene responsible for the genetic defect in Chinese HD patients.

Adult↗

PubMed1998

[A technique of constructing human chromosomal band-specific probe pools using degenerate oligonucleotide primer].

OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.

Chromosome Banding↗

PubMed1998

[Trinucleotide repeats polymorphism of the androgenic receptor gene in the detection of carriers of androgen insensitivity syndrome].

Androgen insensitivity syndrome (AIS) is caused by mutation in androgen receptor (AR) gene. This phenomenon leads to the defect of the virilization of the external genitalia and is one of the causes of human male pseudohermaphroditism. Molecular diagnosis of AR gene mutations is very complex because of heterogeneity of AR mutations. AIS is X-linked recessive trait and therefore the simple diagnosis of AIS carriers is a very important problem. Detection of AIS carriers by using the analysis of CAG repeating sequence polymorphism of exon 1 or AR gene is reported.

Adult↗

PubMed2000

Derivation of a simple microsatellite locus from a compound ancestor in the genus Mus.

Many microsatellite loci contain more than a single repeat motif. These compound microsatellites are perhaps most easily explained as arising from simple ancestors. We have uncovered a contrary example in mice of the genus Mus. Sequence analysis of the locus D1MIT29 in most of the members of the genus Mus reveals that this locus is compound in all species except M. musculus, in which it is simple. Moreover, phylogenetic analyses of base substitutions in the non-repetitive flanking region gives trees which are consistent with the previously accepted phylogenetic hypothesis that M. musculus is nested within the subgenus Mus. This confirms that the history of this locus is similar to that of molecular markers previously used in phylogenetic studies of this group and, therefore, demonstrates that the simple state in this lineage is derived from a compound ancestor. We also demonstrate the utility of this type of nuclear sequence variation for phylogeographic studies in the genus Mus. Finally, our sequences reveal homoplasy for size, reemphasizing the danger of using microsatellite size variation alone when the individuals under study are not closely related.

Animals↗

PubMed1997

A novel method to develop highly specific models for regulatory units detects a new LTR in GenBank which contains a functional promoter.

Functional promoters are composed of individual modules (e.g. transcription factor binding sites, secondary structure elements, repeats) arranged in distinct patterns. Recognition of such patterns is essential for identification of promoters in non-coding sequences. However, this is difficult due to the absence of overall sequence similarity in promoters even if they are regulated in a similar way. We implemented simple formal representations of general features of regulatory regions into an algorithm capable of developing complex models reflecting both the element composition and the functional organization of individual elements (ModelGenerator). Though ModelGenerator requires a very simple initial model (e.g. two modules and their relative order) it will generate a much more sophisticated model by analysis of the training set of at least ten sequences. We show ModelGenerator to successfully model different retroviral long terminal repeat (LTR) classes (Lentivirus as well as avian and mammalian C-type) which contain functional promoters. Database searches with the program ModelInspector demonstrated the high specificity of these models and no apparent false negatives were detected. We also verified one match from GenBank to the mammalian C-type LTR model experimentally and showed this sequence to contain an active promoter. Thus, the concept of modular organization of functional regulatory DNA regions (e.g. promoters) could be successfully implemented into a set of computer tools which might be flexible and specific enough to be suitable for prospective analysis of new genomic DNA sequences.

Algorithms↗

PubMed1986

A variable tandem repeat locus mapped to chromosome band 10q26 is amplified and rearranged in leukocyte DNAs of two cancer patients.

A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation.

Base Sequence↗

PubMed1998

Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes.

Similarities exist among components of simple and complex centromeres that may not have been expected on the basis of wide variation in size and sequence organization of centromeric DNAs among eukaryotes. Support is growing in systems from fungi to Drosophila for a model of centromere assembly and activation that is dependent on a particular underlying chromatin structure but not necessarily on a specific DNA sequence.

Animals↗

PubMed2001

A single, multiplex PCR for differentiating all species of Trichinella.

The genus Trichinella is currently divided into seven species and at least three additional, unclassified genotypes, Trichinella T6, T8 and T9, where both T8 and T9 have been deemed very similar to T. britovi. Other than for the non-encapsulated species, the absence of distinguishing morphological characters and the overlapping nature of the biological characters within this genus make these traits unsuitable for diagnosis. Consequently, we have developed a simple PCR test for the unequivocal differentiation of all currently recognized species of Trichinella including Trichinella T6. DNA sequence data from each Trichinella genotype were generated from internal transcribed spacers, ITS1 and ITS2, and from expansion segment V (ESV) of the rDNA repeat, from which five different PCR primer sets were chosen. When used simultaneously, this primer mix generates a simple and unique electrophoretic DNA banding pattern for each species and genotype. The ESV-derived primer set contributes at least one band to each agarose gel-derived genotypic pattern and therefore functions as an internal control for PCR integrity. Geographical isolates of each Trichinella genotype were used to verify the reliability and reproducibility of respective DNA banding patterns using single muscle larvae.

Animals↗

PubMed1997

Gene transfer using a novel fusion protein, GAL4/invasin.

The delivery of DNA to target cells using simple, defined, nonviral systems has become an area of intense interest in gene therapy. We describe here the development and characterization of one such novel system. A recombinant, bifunctional, fusion protein was expressed and purified from Escherichia coli. This protein consists of the DNA-binding domain of the yeast transcription factor GAL4 fused to the cell binding, internalization domain of the Yersinia pseudotuberculosis inv gene product, invasin. This protein, GAL4/Inv, together with poly-L-lysine, formed complexes with a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains eight repeats of the GAL4 consensus recognition sequence. These complexes were shown to transfect target cells in an invasin receptor-dependent manner, resulting in transient CAT expression. A simple, targeted DNA delivery vehicle, as we describe here, represents a viable approach to nonviral gene delivery.

Adhesins, Bacterial↗

PubMed1999

A fast polymerase chain reaction-mediated strategy for introducing repeat expansions into CAG-repeat containing genes.

We describe a simple method for expanding CAG trinucleotides in CAG-repeat containing genes which, in contrast to other techniques, leaves the original gene sequence intact. Here, we expanded the CAG stretches of a plasmid clone containing the cDNA of the SCA3/MJD gene from 22 CAG up to > 130 CAG repeats using polymerase chain reaction (PCR)-mediated mutagenesis. The method reported in this article requires minimal resources, is very fast, and enables to construct recombinant plasmids carrying large CAG expansions.

Base Sequence↗

PubMed1986

Cloning and characterization of two Onchocerca volvulus repeated DNA sequences.

Two repeated sequences, plasmids pOV8 and pOV26, were cloned and characterized from the filarial parasite Onchocerca volvulus. Both clones can be used to distinguish O. volvulus DNA from other Onchocerca species or other nematodes by restriction fragment length polymorphisms, but neither clone can differentiate between DNA from savanna (Mali) or forest (Ivory Coast) O. volvulus isolates. DNA from one O. volvulus infective larva can be detected by both clones in dot blot hybridization assays. Neither clone cross hybridizes with DNA from host or vector species (human or simuliid, respectively). pOV26 is a member of an interspersed repeated DNA family composed of at least 100 members, and is only observed in the genus Onchocerca. Repeated DNA clone pOV8 cross reacts with DNA from other parasitic filarial nematodes, and is also present in at least 100 copies per O. volvulus genome. pOV26 is a potential tool in the diagnosis of human onchocerciasis, since it is specific for the genus Onchocerca. In the future, we plan to look for regions of these repeated sequences which may serve as a basis for the development of probes to discriminate among Onchocerca species and strains using a simple dot hybridization test.

Animals↗

PubMed1982

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.

Animals↗

PubMed2005

Why do human diversity levels vary at a megabase scale?

Levels of diversity vary across the human genome. This variation is caused by two forces: differences in mutation rates and the differential impact of natural selection. Pertinent to the question of the relative importance of these two forces is the observation that both diversity within species and interspecies divergence increase with recombination rates. This suggests that mutation and recombination are either directly coupled or linked through some third factor. Here, we test these possibilities using the recently generated sequence of the chimpanzee genome and new estimates of human diversity. We find that measures of GC and CpG content, simple-repeat structures, as well as the distance from the centromeres and the telomeres predict diversity as well as divergence. After controlling for these factors, large-scale recombination rates measured from pedigrees are still significant predictors of human diversity and human-chimpanzee divergence. Furthermore, the correlation between human diversity and recombination remains significant even after controlling for human-chimpanzee divergence. Two plausible and non-mutually exclusive explanations are, first, that natural selection has shaped the patterns of diversity seen in humans and, second, that recombination rates across the genome have changed since humans and chimpanzees shared a common ancestor, so that current recombination rates are a better predictor of diversity than of divergence. Because there are indications that recombination rates may have changed rapidly during human evolution, we favor the latter explanation.

Animals↗

PubMed1989

The effectiveness of immediate feedback during the objective structured clinical examination.

Using eight different physical examination or technical stations, 400 examinations were conducted to evaluate the effectiveness of immediate feedback during the Objective Structured Clinical Examination (OSCE). The test group comprised 50 medical students who underwent a standard 4-minute examination followed by 2 minutes of feedback. Immediately following feedback the students repeated an identical 4-minute examination scored by the same examiners. The control group consisted of 50 students from the same class who underwent an identical testing sequence, but instead of receiving feedback, they were instructed to continue their examinations for an additional 2 minutes before repeating the stations. Simple repetition of the task did not significantly improve score (mean increase 2.0%, NS). Extending the testing period from 4 to 6 minutes resulted in a small but significant increase in score (mean 6.7%, P less than 0.001). However, there was a much larger increase in the scores obtained following 2 minutes of immediate feedback compared to pre-feedback performance (mean 26.3%, P less than 0.0001). The majority of students and examiners felt that feedback, as administered in this study, was valuable both as a learning and teaching experience. Short periods of immediate feedback during an OSCE are practical and can improve competency in the performance of criterion-based tasks, at least over the short term. In addition, such feedback provides students with valuable self-assessment that may stimulate further learning.

Clinical Competence↗

PubMed2000

Microsatellite instability correlates with negative expression of estrogen and progesterone receptors in sporadic breast cancer.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments RER+ tumors. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters, including expression of estrogen and progesterone receptors. We analyzed the status of 10 different microsatellite loci (mono- and dinucleotide repeats). In this study, microsatellite size patterns were compared in 88 paired breast-cancer/peripheral-blood DNA samples. Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. MSI in at least two microsatellite markers was observed in 6 out of 88 (7%) of the cases, all belonging to Stage II or III. These MSI data were analyzed using a range of clinicopathological parameters; no correlation between MSI and histopathological characteristics were found. A significant correlation was observed between MSI and negative expression of both estrogen and progesterone receptors (P<0.02), indicating a possible relationship between specific genetic changes at these microsatellite regions and hormonal deregulation in the progression of breast cancer.

Adult↗