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D Sinnett

Publications and source records attributed to D Sinnett.

15 recordsLinked to original sources
PubMed1992

Linkage mapping by simultaneous screening of multiple polymorphic loci using Alu oligonucleotide-directed PCR.

We present the use of our recently described multiple-loci polymorphic DNA markers ("alumorphs") for linkage mapping of the human genome. By using the polymerase chain reaction (PCR) with an Alu-specific primer we could reveal, in a single experiment, up to 20 genomic polymorphisms seen as the presence or absence of amplified DNA fragments originating from genomic segments flanked by Alu repeats. Using this approach we examined genomic DNA samples from two families with a history of pseudovitamin D-deficiency rickets (PDDR), an autosomal recessive disorder. An indication of linkage with the PDDR phenotype was found for one of the polymorphic bands, denoted 30A. A significant linkage [logarithm-of-odds (lod) score greater than 3.0] was obtained between this polymorphism and a number of chromosome 12q markers tightly linked to PDDR. The 30A band specifically hybridized to DNA digests from hybrid cell lines carrying a human chromosome 12, thus independently assigning the 30A marker to this chromosome. Since Alu elements are ubiquitous in human DNA, the use of alternative Alu-specific primers, which reveal different sets of Alu-flanked loci, should provide an efficient and rapid approach to human genetic mapping.

Base Sequence↗
PubMed1992

Alu RNA transcripts in human embryonal carcinoma cells. Model of post-transcriptional selection of master sequences.

Alu master sequences colonized the human genome using RNA as amplification intermediate. To understand this phenomenon better we isolated and analyzed Alu RNA from NTera2D1 pluripotential cells. Northern hybridization, primer extension, cDNA cloning and sequencing data are congruent and demonstrate a low level of Alu specific transcription. These bona fide RNA Polymerase III Alu transcripts, although enriched in the cytoplasm, are not dominated by a single master species but rather originate from a variety of loci. However, when compared with the genomic average, or to repeats from RNA Polymerase II co-transcripts, they belong to the youngest group of Alu subfamilies (p less than 0.001) and have a higher content of intact CpG-dinucleotides. This suggests that Alu transcription is influenced both by mutations and the genomic context, and points to a possible role of DNA methylation in silencing the bulk of genomic repeats. Because of the heterogeneity of Alu transcripts a post-transcriptional selection mechanism recruiting Alu master sequences for retroposition is required. We propose that Alu RNA masters could have evolved as selfish satellites to a more complex retroposition system equipped with a reverse transcriptase activity and that their structure was conserved through "phenotypic" selection of the RNA level.

Base Sequence↗
PubMed1992

Single-strand conformational polymorphisms (SSCP): detection of useful polymorphisms at the dystrophin locus.

We searched for DNA polymorphisms in seven amplified fragments of the dystrophin gene. Three fragments exhibited variable mobilities during nondenaturing strand-separating gel electrophoresis (SSGE). These variants were due to single base changes (three transversions and one transition). Three were intronic (upstream from exons 17, 15, and 48) and one was in exon 48. The frequencies of these sequence variants were determined in a sample of 54 normal X chromosomes of Caucasian origin. One of these DNA polymorphisms was observed in every 650 bp tested and the average heterozygosity was 0.05% per base pair (0.08% if exons were excluded). Such a detection density and the fact that single-strand conformational polymorphisms do not depend on the presence of any specific sequence makes them especially valuable as genetic markers. In the dystrophin locus this approach could allow simultaneous detection of frequent deletions.

DNA, Single-Stranded↗
PubMed1991

Alu RNA secondary structure consists of two independent 7 SL RNA-like folding units.

The amplification of genomic Alu elements by retroposition, i.e. by reintegration of reverse-transcribed RNA, suggests that Alu RNA plays an important role in this process. We report enzymatic studies of the secondary structure of Alu RNAs transcribed in vitro from two recently retroposed Alu elements. These experiments show that the dimeric organization of an Alu sequence is reflected in its RNA folding. Alu subunits fold independently, conserving secondary structure motifs of their progenitor 7 SL RNA molecule. Energy minimization analysis indicates that this folding pattern is also characteristic of different Alu and Alu-like sequences and has been conserved since primate divergence. By analogy to 7 SL RNA, the Alu RNA folding may be important for specific interactions with proteins. This could indicate a physiological function for Alu transcripts. However, this can be also seen as a structural adaptation leading to efficient retroposition of these sequence elements.

Base Sequence↗
PubMed1991

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved.

In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.

Animals↗
PubMed1991

G proteins in normal rat pituitaries and in prolactin-secreting rat pituitary tumors.

It is still undetermined which GTP-binding (G) protein is involved in the regulation of prolactin (PRL) release and through which effector. This study shows that, when compared to normal pituitary tissue, the levels of alpha o protein were very low in dopamine (DA)-resistant, PRL-secreting pituitary tumors 7315a and MtTW15, while alpha o mRNA was present in the two tumors. In the MtTW15 tumor alpha i1, alpha i2 and alpha i3 levels were decreased while those of alpha s42 and alpha s47 were increased, and in the 7315a tumor alpha i2, alpha i3 and beta levels were decreased and those of alpha s47 increased. In an estrone-induced, DA-sensitive prolactinoma the levels of alpha i3 were greatly reduced. DA was unable to inhibit basal PRL release by 7315a and MtTW15 and basal cAMP accumulation by adenomatous and MtTW15 cells. Vasoactive intestinal peptide (VIP) increased both cAMP accumulation and PRL release by all cell preparations which could be suppressed by DA with adenomatous and 7315a but not with MtTW15 cells. These and previously published results provide circumstantial evidence that alpha o, alpha i1 and alpha i3 are all involved in the transduction of the DA inhibitory message while alpha s47 transduces cAMP activating messages and alpha s42 is responsible for the constitutive activation of L-type Ca2+ channels, adenylate cyclase and baseline PRL release.

Adenoma↗
PubMed1990

Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition.

Base Sequence↗
PubMed1990

Alumorphs--human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers.

The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.

Base Sequence↗
PubMed1989

The gene for incontinentia pigmenti is assigned to Xq28.

A linkage study of eight families with incontinentia pigmenti (IP) has been performed, and linkage to site DXS52 has been established. We suggest that the IP locus lies in the Xq terminal region on the long arm of the X chromosome.

Chromosome Mapping↗
PubMed1989

High intensity focused ultrasound--a surgical technique for the treatment of discrete liver tumours.

The treatment of discrete liver tumours is often a difficult clinical problem. High intensity, focused ultrasound may provide one form of therapy for such disease. The ability to focus ultrasound precisely on a predetermined volume allows the possibility of selective tissue destruction at this position without damage to intervening tissues. We have investigated this both in vivo and in excised liver samples in vitro. Quantitative and qualitative studies have been carried out on the relationship between the ultrasonic exposure and the lesion shape, position and volume. In addition, the highly echogenic nature of the ultrasonic lesion has been studied, in an attempt to determine whether 'real time' observation of the extent of tissue damage is feasible.

Animals↗
PubMed1988

Lesch-Nyhan syndrome: molecular investigation of three French Canadian families using a hypoxanthine-guanine phosphoribosyltransferase cDNA probe.

Using human hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA and an anonymous probe 36B-2, we examined the segregation of restriction fragment length polymorphism (RFLP) alleles with the Lesch-Nyhan phenotype in three affected families. Two families were informative. Five carriers of the mutation in one family and two potential carriers in the second were heterozygous for either one or both polymorphisms allowing for prenatal diagnosis. Southern blot patterns in patients from these three families indicated the absence of major structural alterations in the defective gene. Northern analysis using HPRT cDNA as a probe revealed no hybridizing RNA in one patient, whereas normal size mRNA was expressed at a very low level in the second and at a level comparable to normal in the third. These data are consistent with heterogeneity of Lesch-Nyhan genetic lesions resulting from point mutations or small DNA deletions or rearrangements, which may affect transcription, stability, or integrity of the HPRT message.

Blotting, Northern↗
PubMed1988

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11.

Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster X human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter - (DXS7, DXS146, DXS255) - IP1 - (DXS14, DXS90) - DXS106 - qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.

Animals↗