Papers by Belinda Rossiter
Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, Oct 31, 1992
Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) ... more Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TG R) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented.
The Polymerase Chain Reaction, 1994
The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) g... more The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1990
DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiatio... more DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1992
Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) ... more Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TG R) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1990
CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation... more CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.
Faseb Journal - FASEB J, 1991
A wide variety of techniques are available for detecting disease-causing mutations within human g... more A wide variety of techniques are available for detecting disease-causing mutations within human genes; this report provides a brief review of such procedures. Good communication and exchange of materials between the clinical genetics field and the Human Genome Initiative will benefit both.
Somatic Cell and Molecular Genetics, 1990
There are many reports of antisense inhibition of gene expression in cultured cells. We have gene... more There are many reports of antisense inhibition of gene expression in cultured cells. We have generated four strains of transgenic mice expressing antisense hypoxanthine guanine phosphoribosyltransferase (HPRT) RNA in brain, or heart and liver, or all three organs. In the brain of one strain, the level of antisense RNA in the different brain regions roughly correlates with the degree of inhibition of the native HPRT mRNA in those same regions. Despite this decrease of up to 60% of endogenous HPRT mRNA, no reproducible reduction in HPRT activity has been observed. Possible reasons for the differences between the effectiveness of antisense inhibition in cultured cells and transgenic animals are discussed.
Obstetrical & Gynecological Survey, 1993
Mutagenesis, 1990
The Chinese hamster hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient cell line TG1... more The Chinese hamster hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient cell line TG15 produces apparently normal HPRT mRNA by northern analysis and was therefore presumed to contain a point mutation within the coding region. Sequencing cDNA from the TG15 cell line revealed an A to G transition which results in the substitution of the amino acid glycine for aspartic acid at position 135. TG15 cells revert to wild-type HPRT activity upon exposure to monofunctional alkylating agents. A rapid test to assay the site of the TG15 point mutation has been developed, utilizing the polymerase chain reaction and allele-specific oligonucleotide screening. In all revertants studied, the original point mutation has been corrected to the wild-type sequence. The TG15 point mutation lies within a proposed catalytic domain of the HPRT protein in common with other phosphoribosyltransferases.
Mutagenesis, 1988
The revertibility of three spontaneous hypoxanthine phosphoribosyl transferase (HPRT)-deficient V... more The revertibility of three spontaneous hypoxanthine phosphoribosyl transferase (HPRT)-deficient V79 cell lines has been determined after exposure to a number of alkylating agents. TG11 and 19 reverted at frequencies ranging from 1 X 10(-5) to 1 X 10(-4) after exposure to doses of ethylmethane sulphonate (EMS) N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) resulting in surviving fractions between 1.0 and 0.1. Reversion frequencies in TG15 ranged from 10(-7) to 5 x 10(-6) over a similar dose range. The relative efficiencies of different monofunctional alkylating agents in causing reversion of TG11 at equitoxic doses were ENU greater than EMS greater than N-ethyl-N-nitroso-guanidine greater than MNU greater than N-methyl-N-nitrosoguanidine greater than methylmethane sulphonate. Revertant frequencies for all three cell lines were maximal immediately after treatment and declined thereafter at a rate inversely proportional to dose. Such kinetics are explicable if reversion is due to miscoding opposite alkylated guanines. Reversion frequencies after N-butyl-N-nitrosourea exposure were 100-fold lower than after MNU and kinetics of expression of revertant colonies differed. Frequencies were low immediately after treatment, increased between 0 and 24 h then remained at a plateau. Similar kinetics were observed after chlorozotocin and bis-chloroethylnitrosourea exposure. This difference in expression kinetics suggests that reversion in this case is not the result of direct miscoding but of errors in excision repair. TG11, 15 and 19 had low spontaneous mutant frequencies which were either unaffected or only marginally increased by treatment with 5-azacytidine.(ABSTRACT TRUNCATED AT 250 WORDS)
International Journal of Technology Assessment in Health Care, 1994
Several routine procedures are available for diagnosis of diseases caused by an alteration in a s... more Several routine procedures are available for diagnosis of diseases caused by an alteration in a single gene. These techniques include Southern analysis, the polymerase chain reaction, allele-specific oligonucleotide screening, automated DNA nucleotide sequencing, and linkage analysis. DNA testing procedures can be used for diagnosis of disease, determination of carrier status in affected families, or general screening of the population. Some of the more commonly used techniques and their applications are described in this article.
Genomics, 1991
The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) g... more The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.
Drugs & Aging, 1995
As the Human Genome Project gathers speed, new disease genes are rapidly being found. Important a... more As the Human Genome Project gathers speed, new disease genes are rapidly being found. Important as these discoveries are, they are only the beginning of the process of characterising, diagnosing and treating genetic diseases. We now have the potential to predict the onset of many disorders before the appearance of clinical symptoms, even though treatment is not always available. In this review we have used a number of examples to illustrate various aspects of the presymptomatic diagnosis of genetic disease and, where possible, late-onset disorders have been chosen as examples. When treatment is available, the diagnosis of a disease before appearance of symptoms can greatly improve the prognosis. When treatment is not available, reasons to undergo presymptomatic testing may not be so obvious. However, appropriate lifestyle changes or medical surveillance can sometimes delay onset or decrease severity of a disorder. Even if no treatment is available, genetic testing and counselling for the patient and family members can provide useful information for future planning.
Clinical Obstetrics and Gynecology, 1993
Teratogenesis, Carcinogenesis, and Mutagenesis, 1989
We have developed a rapid screening method using the polymerase chain reaction (PCR) for detectin... more We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light-and X-ray-induced hprtdeficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (1 1/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.
Annals of Surgical Oncology, 1995
The Human Genome Project is a coordinated effort to define the human genetic blueprint. The goals... more The Human Genome Project is a coordinated effort to define the human genetic blueprint. The goals include construction of a variety of maps of the human genome, including the identification and localization of all genes. The discovery of genes responsible for human diseases has had a significant impact on the practice of medicine. Methods for defining the human genome include cytogenetic, physical, and genetic mapping techniques. A variety of strategies have been used to identify human genes, especially those genes that are responsible for disease. Once a disease gene has been identified, this information can be used to develop new diagnostic and therapeutic procedures. A number of disease genes have already been identified, leading to improved diagnosis and novel approaches to therapy. A new type of mutation, trinucleotide repeat expansion, has been found to be responsible for at least seven diseases with an unusual inheritance pattern. Materials and technology generated by the Human Genome Project and related research have provided important tools for the diagnosis and treatment of patients afflicted with genetic diseases.
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Papers by Belinda Rossiter