PubMed Health⌕ Search

Biomedical subjects

E M Simpson

Publications and source records attributed to E M Simpson.

36 records · Page 2Linked to original sources
PubMed1997

Expression of hygR in transgenic mice causes resistance to toxic effects of hygromycin B in vivo.

Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concern. In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of prokaryotic genes of antibiotic resistance in vivo. We characterized the acute and tissue-specific toxicity of hygromycin B in transgenic mice bearing the hygromycin B phosphotransferase (hygR) gene under control of a constitutive promoter. We characterized the tissue-specific expression of hygR mRNA and also investigated the acute toxicity of hygromycin B in hygR and wild-type mice. The hygR mRNA reached its highest levels in brain and reached intermediate levels in spleen, muscle, kidney, liver and testis. The lowest levels were detected in heart and lungs. The hygR expression in transgenic animals caused an 89-fold increase in the approximate lethal dose of hygromycin B compared with wild-type mice. Serum biochemical analysis of hygR and wild-type mice treated with lethal doses of hygromycin B indicated liver and kidney damage measured as ALT, AST and BUN. On the morphological level, these changes led to acute tubular nephrosis in wild-type mice and acute liver damage in hygR mice. Our results show that constitutive expression of the bacterial hygR gene in transgenic mice in vivo confers resistance to hygromycin B.

Animals↗
PubMed1996

Mouse Y-specific repeats isolated by whole chromosome representational difference analysis.

Representational difference analysis (RDA) was used to generate Y-specific probes by enriching for and cloning the differences between the male (XY) and the female (XX) C57BL/6J mouse genomes. Characterization of 35 clones revealed 12 families related by sequence similarity. One clone from each family was chosen for detailed analysis by Southern blot hybridization, polymerase chain reaction (PCR) on normal and aberrant genomes (Sxr), and fluorescence in situ hybridization. From one difference product we have characterized 12 Y-specific probes for hybridization, created seven male-specific PCR assays, mapped all repeat families, and identified one repeat with a distinct XY homology. We report the first cloning of a Y-specific long interspersed repeat element (LINE) fragment. In total, RDA has identified six novel Y Chromosome repeat families and allowed us to extend the characterization of six known Y repeats. We conclude that this novel use of RDA for whole chromosome subtraction successfully enriches chromosome-specific sequences and is suitable for the rapid generation of new Y Chromosome-specific probes.

Animals↗
PubMed1996

Frequencies of cystic fibrosis mutations in the Maine population: high proportion of unknown alleles in individuals of French-Canadian ancestry.

Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders in Caucasian populations. A mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes this disorder. Reported here is the first analysis of CF mutations in the Maine population. We have screened 263 CF chromosomes for 16 previously reported mutations. Analysis of DNA from 124 apparently unrelated CF patients and 15 obligate carrier parents (whose partner and affected child were unavailable for study) resulted in the identification of 91% of the CF alleles and complete genotyping of 85% of the patients. The frequencies (%) of these mutations in the Maine population are delta F508 (75% of the chromosomes), G85E (0.76), R117H (0.76), I148T (1.1), 621 + 1G --> T (1.1), 711 + 1G --> T (3.0), A455E (1.1), 1717-1G --> A (1.1), G542X (1.9), G551D (1.9), R560T (0.76), Y1092X (0.38), W1282X (0.38), and N1303K (1.5). The exon 10 mutation, delta I507, and the exon 11 mutation, R553X, were not observed. Surprisingly, whereas only 5% of the alleles remain unidentified in the non-French population, the unidentified proportion in the French population is 19%. CF testing for the Maine population will be further improved as the as yet unidentified CF mutations in this population are characterized.

Alleles↗
PubMed1995

Chromosomal assignment of the genes for proprotein convertases PC4, PC5, and PACE 4 in mouse and human.

The genes for three subtilisin/kexin-like proprotein convertases, PC4, PC5, and PACE4, were mapped in the mouse by RFLP analysis of a DNA panel from a (C57BL/6JEi x SPRET/Ei)F1 x SPRET/Ei backcross. The chromosomal locations of the human homologs were determined by Southern blot analysis of a DNA panel from human-rodent somatic cell hybrids, most of which contained a single human chromosome each. The gene for PC4 (Pcsk4 locus) mapped to mouse chromosome 10, close to the Adn (adipsin, a serine protease) locus and near the Amh (anti-müllerian hormone) locus; in human, the gene was localized to chromosome 19. The gene for PC5 (Pcsk5 locus) mapped to mouse chromosome 19 close to the Lpc1 (lipocortin-1) locus and, in human, was localized to chromosome 9. The gene for PACE4 (Pcsk6 locus) mapped to mouse chromosome 7, at a distance of 13 cM from the Pcsk3 locus, which specifies furin, another member of this family of enzymes previously mapped to this chromosome. This is in concordance with the known close proximity of these two loci in the homologous region on human chromosome 15q25-qter. Pcsk3 and Pcsk6 mapped to a region of mouse chromosome 7 that has been associated cytogenetically with postnatal lethality in maternal disomy, suggesting that these genes might be candidates for imprinting.

Animals↗
PubMed1994

Expression of a mouse Zfy-1/lacZ transgene in the somatic cells of the embryonic gonad and germ cells of the adult testis.

The Zfy-1 and Zfy-2 genes, which arose by gene duplication, map to the mouse Y chromosome and encode nearly identical zinc-finger proteins. Zfy-1 is expressed in the genital ridge and adult testis and likely encodes a transcription activator. Although potential roles in sex determination and spermatogenesis have been hotly debated, the biological functions of Zfy-1 remain unknown. To study the gene's regulation, transgenes with 21-28 kb of Zfy-1 5' flanking DNA placed upstream of lacZ were constructed in plasmids or created by homologous recombination of coinjected DNA molecules. The resulting transgenic mice expressed beta-galactosidase in the genital ridge of both males and females starting between embryonic day 10 and 11 (E10-E11), peaking at E12-E13 and then declining to low levels by E15, a pattern that matches Zfy-1 mRNA as detected by RT-PCR. This lacZ expression in genital ridge was confined to somatic cells as demonstrated by its absence from the alkaline phosphatase-positive germ cells. It had been reported previously that Zfy-1 mRNA was absent from the embryonic gonad of homozygous W(e) embryos, which virtually lack germ cells. By contrast, we observed normal expression of the Zfy-1/lacZ transgene when introduced into the W(e) background, suggesting that germ cells are not necessary for expression. In the adult, the Zfy-1/lacZ transgene is expressed abundantly in developing germ cells. Extragonadal (kidney, meninges, arteries, choroid plexus) expression of the transgene was also observed in embryos. A smaller transgene with only 4.3 kb of Zfy-1 5' flanking DNA was expressed only in germ cells of adult mice. These results suggest that an enhancer for germ cell expression in the adult lies near the Zfy-1 promoter and that an enhancer for expression in the somatic cells of the embryonic gonad is located further 5'.

Alkaline Phosphatase↗
PubMed1991

An interstitial deletion in mouse Y chromosomal DNA created a transcribed Zfy fusion gene.

The small portion of the mouse Y chromosome retained in the Sxra transposition is thought to carry at least five genes including, as demonstrated here, the entirety of the zinc-finger genes Zfy-1 and Zfy-2. Sxrb, a derivative of Sxra, was previously thought to retain Zfy-1 but to be deleted for Zfy-2. Here we show that Sxrb differs from Sxra as the result of unequal crossing-over between Zfy-1 and Zfy-2. This unequal crossing-over created a transcribed Zfy-2/1 fusion gene and an interstitial deletion. Our data and previous results together suggest that this deletion encompassed the 3' portion of Zfy-2, the histocompatibility gene Hya, the spermatogenesis factor Spy, and the 5' portion of Zfy-1. We suggest that not only Zfy but also other neighboring genes such as Spy and Hya may exist in two copies on the Y as the result of a large tandem duplication during rodent evolution.

Animals↗
PubMed1990

Chromosomal localization of ZFX--a human gene that escapes X inactivation--and its murine homologs.

The ZFY gene, found in the sex-determining region of the human Y chromosome, encodes a zinc-finger protein that may be the pivotal sex-determining signal. A closely related gene, ZFX, is found on the human X chromosome, and it may also function in sex determination. ZFX is one of a few genes on the human X chromosome that are known to escape X inactivation. We report the localization of ZFX, by meiotic linkage analysis and physical mapping, distal to POLA but proximal to DXS41 (p99-6), near the boundary of bands Xp21.3 and Xp22.1. (Our results suggest the following order of loci in Xp21-p22: cen-DMD-[GK,AHC]-DXS67 (pB24)-POLA-ZFX-[DXS41 (p99-6), DXS274 (CRI-L1391)]-DXS43 (pD2)-pter.) These findings contradict the model that escape from X inactivation is limited to genes near the short-arm telomere (i.e., in Xp22.3). Instead, escape from X inactivation is likely a property of several noncontiguous segments of the X chromosome. Curiously, in mouse, the homologous Zfx gene maps to X chromosome band D, near the center from which an X-inactivating signal is thought to spread. As judged by comparative mapping, it appears that an X-chromosomal segment that spans the ZFX and DMD genes has remained grossly intact during the divergence of mouse and human from a common ancestor. Conservation of this chromosomal segment may extent to marsupials, where homologs of the ZFX and DMD genes have been observed in proximity, but on an autosome. While autosomal homologs of ZFX have not been observed in other placental mammals, a locus derived from a processed Zfx transcript is found on mouse chromosome 10 band B3 or B4.

Animals↗
PubMed1990

Mouse Zfx protein is similar to Zfy-2: each contains an acidic activating domain and 13 zinc fingers.

The Zfy gene is located on the Y chromosome of placental mammals and encodes a zinc finger protein which may serve as the primary sex-determining signal. A related gene, Zfx, is similarly conserved on the X chromosome. Unlike that in most mammals, the mouse genome contains four homologous zinc finger loci: Zfy-1, Zfy-2, Zfx, and Zfa (on an autosome). We report that, in contrast to the mouse Zfy genes, Zfx is widely transcribed in embryos, newborns, and adults, both male and female. Moreover, Zfx transcripts contain long 3' untranslated sequences which are phylogenetically conserved. Zfa is a processed gene derived from Zfx. An analysis of cDNA clones demonstrated that Zfx encodes a 799-amino-acid protein that is 70% identical to the mouse Zfy-1 and Zfy-2 proteins. Zfx, Zfy-1, and Zfy-2 contain highly acidic amino-terminal domains and carboxy-terminal regions containing 13 zinc fingers. When fused to the DNA-binding domain of GAL4, the acidic domains of Zfx and Zfy-2 activated transcription in yeast cells.

Amino Acid Sequence↗
PubMed1989

Nystagmus after medulloblastoma.

Ten survivors of medulloblastoma were examined for residual opthalmological signs. Gaze-evoked nystagmus occurred in six children, four of whom also had pronounced upbeat nystagmus. This is a rare and specific type of nystagmus associated with lesions of the cerebellar vermis and medulla, and has not been described previously as a long-term sequel of medulloblastoma. The recognition of upbeat nystagmus is important because visual acuity may be reduced in upward gaze and difficulties at school may ensue for these children, who may be already intellectually and behaviourally compromised.

Cerebellar Neoplasms↗
PubMed1988

Bacteraemia related to indwelling central venous catheters: prevention, diagnosis and treatment.

Infective episodes in immunocompromised children with indwelling central venous catheters were studied prospectively for one year. Culture of catheter hubs and skin at catheter entry sites during the first six months suggested that hub contamination was important in the pathogenesis of catheter colonization. The incidence of catheter-related bacteraemia, and possible catheter-related bacteraemia, fell by 56.5% following alterations in the protocol for manipulative care of catheters, from 5.82 per 1000 catheter days in the first six months to 2.53 per 1000 catheter days in the subsequent six months. A firm diagnosis of catheter-related bacteraemia was made simply and economically by a pour-plate quantitative blood culture technique. Attempts at eradication of catheter-related bacteraemia without removal of the catheter were successful in all cases.

Adolescent↗
PubMed1987

The sex-determining region of the human Y chromosome encodes a finger protein.

The presence or absence of the Y chromosome determines whether a mammalian embryo develops as a male or female. In humans, genetic deletion analysis of "sex-reversed" individuals has identified a small portion of the Y chromosome necessary and sufficient to induce testicular differentiation of the bipotential gonad. We report the cloning of a 230-kilobase segment of the human Y chromosome that contains some or all of the testis-determining factor gene (TDF), the master sex-determining locus. The cloned region spans the deletion in a female who carries all but 160 kilobases of the Y. Certain DNA sequences within this region were highly conserved during evolution; homologs occur on the Y chromosomes of all mammals examined. In particular, homologous sequences are found within the sex-determining region of the mouse Y chromosome. The nucleotide sequence of this conserved DNA on the human Y chromosome suggests that it encodes a protein with multiple "finger" domains, as first described in frog transcription factor IIIA. The encoded protein probably binds to nucleic acids in a sequence-specific manner, and may regulate transcription. Very similar DNA sequences occur on the X chromosome of humans and other mammals. We discuss the possibility that the Y-encoded finger protein is the testis-determining factor, and propose models of sex determination accommodating the finding of a related locus on the X chromosome. The presence of similar sequences in birds suggests a possible role not only in the XX/XY sex determination system of mammals, but also in the ZZ/ZW system of birds.

Amino Acid Sequence↗
PubMed1987

Hybrid leukaemia of T cell and myeloid lineages: cytogenetic distinction from second (induced) malignancy.

The therapeutic and prognostic implications of relapse and clonal evolution of leukaemia are substantially different from those of secondary (induced) malignancy. This report documents the case of a patient who presented with apparent acute non-lymphocytic leukaemia (ANLL) following therapy for acute (T-cell) lymphoblastic leukaemia (ALL) 4 years previously. Morphologically and cytochemically the cells were of myeloid type, but the cell markers showed a T cell lineage. Cytogenetic studies confirmed that this was a relapse of T cell ALL with a phenotypic change, rather than a second malignancy induced by chemotherapy. A 14q deletion present at initial diagnosis recurred at relapse, with the addition of cells with complete deletion of chromosome 14, indicating clonal evolution.

Child↗
PubMed1987

Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.

Amino Acid Sequence↗