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A J Grosovsky

Publications and source records attributed to A J Grosovsky.

At least 37 records · Page 2Linked to original sources
PubMed1995

Mutational spectrum of X-ray induced TK- human cell mutants.

Mutations induced by ionizing radiation have historically elicited significant public concern. However, only a limited database of ionizing radiation-induced point mutations is available, particularly at endogenous human cell loci. Here, we report the mutational spectrum for 184 X-ray induced TK- mutants derived from TK6 human lymphoblasts. This report represents the first large scale utilization of the tk locus for investigation of mutational specificity at the DNA sequence level. Rapid, single nucleotide sequencing assays at frameshift polymorphism sites in tk exons 4 and 7 were used to partition TK- mutants into two groups: 126 were attributed to either partial gene deletion or to loss of heterozygosity, and DNA sequence alterations were identified for 51. X-ray-induced point mutations included all classes of transitions and transversions, tandem base substitutions, frameshifts, small deletions and a small duplication. The distribution within tk was characterized by clustering at some sites. Twelve TK- point mutations, including five entirely within the coding sequence in exons 3 and 4, resulted in aberrant splicing of the tk transcript. The spectrum of X-ray-induced point mutations was found to be highly reproducible when TK- mutations were compared with HPRT- mutations in TK6. A statistically significant decrease in transitions (P = 0.04) was observed in the combined data set as compared to the spontaneous background. These findings suggest a reproducible pattern which may be utilized in recognizing radiation-induced mutations at other loci of interest.

Alleles↗
PubMed1995

Extensive loss of heterozygosity accounts for differential mutation rate on chromosome 17q in human lymphoblasts.

In order to investigate the influence of loss of heterozygosity (LOH) events on mutation rate, we studied two closely related human lymphoblastoid cell lines, AHH-1 (h2E1.v2) and MCL-5, which are heterozygous at the tk locus (chromosome 17q23-25). Although they have similar mutant fractions at the hprt locus, the mutant fraction and rate at tk is four to five times higher in AHH-1. Analysis of 58 spontaneous TK- mutants from AHH-1 and MCL-5 showed that the occurrence of LOH events was more frequent (23/24) in AHH-1 than MCL-5 (16/34). A set of five microsatellite polymorphism loci was used to map the extent of LOH along chromosome 17q. In AHH-1 cells, 15/23 of the LOH events encompassed at least 35% of the sex-averaged genetic length of chromosome 17q (98 cM). Additionally, the next most extensive category of LOH accounted for 5/23 TK- mutants, and encompassed at least 17 cM. In contrast, LOH events observed in MCL-5 are very restricted in extent; only one LOH tract extended as far as 4 cM from tk. The higher mutation rate at tk in AHH-1 can, therefore, be entirely attributed to the recovery of chromosomal scale LOH in viable, normal growth TK- mutants. Furthermore, these data demonstrate that the regional potential for LOH is likely to be an important determinant of mutation rate for loci within that chromosomal segment.

Aminacrine↗
PubMed1995

Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines.

We have examined the extent of HPRT- total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT- deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (> 1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT- total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT- large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions.

B-Lymphocytes↗
PubMed1994

An abundantly expressed hemolymph glycoprotein isolated from newly parasitized Manduca sexta larvae is a polydnavirus gene product.

Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp C. congregata. CcPDV virions are present in large numbers in the oviducts of C. congregata and injected with eggs into the hemocoel of M. sexta larvae during parasitization. Injection of sucrose density purified virions into nonparasitized larvae causes several of the parasitism-induced alterations in the physiology of host larvae that occur as a result of natural parasitism, including the synthesis of novel hemolymph proteins and abrogation of the host's immune response against the developing parasites. One of these proteins, early-expressed protein 1 (EP1), is a 190-kDa molecule which constitutes up to 5% of the total hemolymph protein by 24 hr following oviposition by the wasp. Using N-terminal sequence data for EP1 to construct primers for use in the polymerase chain reaction, we amplified and cloned a cDNA corresponding to the gene encoding EP1. This cDNA hybridized to DNA of the CcPDV genome, but not to DNA isolated from M. sexta larvae, suggesting that EP1 is a CcPDV gene product. A cDNA clone was isolated from an expression library generated from RNA extracted from newly parasitized M. sexta larvae. Sequence analysis of the cDNA clone revealed the presence of an open reading frame of 819 bp encoding a protein of 30.7 kDa. In vitro transcription/translation of the cDNA clone produced a protein of approximately 31 kDa, which was immunoprecipitated by EP1-specific polyclonal antiserum generated against purified deglycosylated EP1. EP1-like sequences also were amplified from male wasp genomic DNA, suggestive of integration of EP1-like sequences in the genome. This report constitutes the first evidence that a specific protein isolated from a parasitized host insect is a wasp polydnavirus gene product.

Amino Acid Sequence↗
PubMed1994

Spectrum of X-ray-induced mutations in the human hprt gene.

We have characterized the molecular spectrum of mutations in 116 X-ray-induced and 78 spontaneous, HPRT- mutants derived from the human B lymphoblastoid cell line TK6. Multiplex PCR analysis demonstrated that the overall representation of large deletions was not significantly different in the two spectra. However, highly significant differences were observed for specific deletion types. Total gene deletions represented 41/78 (0.53) X-ray-induced, but only 7/43 (0.16) spontaneous deletions (P < 0.0001). In contrast, 5' terminal deletions were significantly more common among spontaneous (17/43, 0.40) than X-ray-induced (13/78, 0.17) large deletions (P = 0.0079). The types of point mutations induced by X-ray exposure were very diverse including all classes of transitions and transversions, tandem base substitutions, frameshifts, small deletions and a deletion/insertion compound mutation. Compared to spontaneous data, radiation-induced point mutations exhibited a reduced number of transitions and an increased representation of small deletions. Small deletions were uniformly surrounded by direct sequence repeats. The distribution of point mutations was characterized by a cluster within the 5' portion of exon 8. Thirteen HPRT- point mutations exhibited aberrant splicing. Four of these were attributable to coding sequence alterations in exons 4 and 8. These results suggest that it may be possible to identify hallmark mutations associated with X-ray exposure of human cells.

B-Lymphocytes↗
PubMed1993

Spectrum of spontaneous HPRT- mutations in TK6 human lymphoblasts.

The occurrence of deletions, coding sequence alterations, and intronic changes leading to aberrant splicing has been characterized among 33 spontaneous HPRT- mutants in TK6 human lymphoblasts. Deletions detectable by multiplex PCR amplification accounted for 45% (15/33) of the mutant collection. Base substitutions represented 30% (10/33) of the total, and were predominated by changes at G:C base pairs. The remaining mutants were distributed among frameshifts (9%, 3/33), small deletions (6%, 2/33), and compound alterations (9%, 3/33). Five mutants (15%) demonstrated aberrant splicing of the hprt transcript. A cluster of 4 deletion/insertion events was identified in hprt exon 6. A nearly perfect 13 bp duplication differed from the original sequence only by an A:T to G:C transition, which was observed as a unique alteration in another HPRT- mutant. A model involving correction of a mismatch in a secondary structure formed by the duplicated sequence may account for these results.

B-Lymphocytes↗
PubMed1993

Genetic instability on chromosome 16 in a human B lymphoblastoid cell line.

Mutagenesis at the aprt locus in TK6 human lymphoblasts has been found to occur at an unusually high rate (1.2 x 10(-9)) for a homozygous diploid locus. Evaluation of linked microsatellite polymorphisms demonstrated that loss of heterozygosity (LOH) accompanies conventional intragenic sequence alterations in each APRT- mutant. LOH occurred without allele preference. The extent of loss was highly uniform, ranging from 16q12 to 16qter in 36/38 APRT- mutants. Fluorescence in situ hybridization (FISH), used in conjunction with microsatellite analysis, demonstrated that the loss was not attributable to physical deletion, nondisjunction, or nondisjunction with reduplication of the remaining chromosome. LOH thus appears to be recombinationally mediated. FISH analysis also detected translocations affecting chromosome 16 in 4/20 APRT- mutants examined. APRT- mutants appear to arise as part of a genetic instability phenomenon since three distinct genetic alterations affecting chromosome 16 are recovered in single clones at a detectable rate. These events may be mechanistically related to early events in gene amplification.

Adenine Phosphoribosyltransferase↗
PubMed1993

DNA-sequence specificity of mutations at the human thymidine kinase locus.

We have established a system for the study of DNA-sequence specificity at a functionally heterozygous thymidine kinase (tk) locus in a human lymphoblastoid cell line (TK6). Characterization of the parental locus demonstrated that the 2 tk alleles were fortuitously distinguished by differential gene expression. One round of PCR amplification yielded a specific tk cDNA product only for the functional parental allele. Analysis of cDNA from newly mutated alleles which retain substantial levels of expression is thus simplified. Amplification and sequencing of tk genomic sequences was used for analysis of low expression mutants, and in order to distinguish and characterize deletion and splicing mutations. DNA-sequence analysis of the parental locus identified a frameshift in tk exon 4 of the non-functional parental allele, and surprisingly, an exon 7 frameshift mutation in the functional tk allele. This exon 7 frameshift results in a predicted alteration of the final 21 amino acids of the TK protein, and a C-terminal extension of 131 additional amino acids. Since TK6 is phenotypically TK+, we can infer that this major C-terminal modification does not eliminate enzymatic activity. The system was utilized for the analysis of 36 spontaneous TK- mutants. Loss of heterozygosity accounted for 58% of the mutations, 11% were attributable to intragenic deletions, and the remainder involved point mutations, primarily G:C to A:T transitions.

Amino Acid Sequence↗
PubMed1993

Evidence for high-frequency allele loss at the aprt locus in TK6 human lymphoblasts.

The aprt locus in TK6 human lymphoblasts has been previously shown to have an unusual mutation frequency (5 x 10(-8) and a gene dosage of 2. Measurements of mutation rate (1.2 x 10(-9)) reported here, confirm the mutation-frequency observations. These results are not easily accommodated by models of the gene as functionally homozygous or heterozygous. Characterization of all exon and intron sequences identified no polymorphism which could distinguish two heterozygous aprt alleles. Furthermore, autoradiographs of 16 spontaneous APRT- mutants demonstrate a variety of unique sequences. If aprt were heterozygous, wild-type sequence from the alternate allele would be observed at positions where mutations have occurred. These observations cannot be explained by conventional loss of heterozygosity in which the same mutated allele would be repeatedly recovered. We therefore propose that aprt is a functionally homozygous locus. APRT- mutants may arise by conventional mutation of one allele, and high-frequency loss or conversion of the alternate allele.

Adenine Phosphoribosyltransferase↗
PubMed1993

Insertional inactivation of the tk locus in a human B lymphoblastoid cell line by a retroviral shuttle vector.

Insertional mutagenesis represents an inherent risk in retrovirally mediated gene therapy, but it may be a useful experimental strategy for identification and isolation of novel cellular loci. In this investigation we have established a model system using a heterozygous thymidine kinase (tk) marker locus in a human B lymphoblastoid cell line, and a M-MuLV based shuttle vector. The frequency of TK- mutants in cells carrying 1-2 proviruses per genome is approximately 2 x 10(-5), a 5-fold increase as compared to an uninfected control population. Southern analysis of a set of 13 retrovirus infected TK- mutants revealed a predominance of rearrangements among those mutants which had not undergone loss of heterozygosity. No consistent relationship was found to exist between the occurrence of a rearrangement and tk gene expression as detected by northern analysis. The mechanisms of retroviral shuttle vector insertional mutagenesis were characterized in more detail by focusing on a single TK- mutant, T2. The single proviral insert in T2 was found to lie within tk intron 2, in parallel orientation to the direction of tk transcription. DNA sequence analysis of tk cDNA revealed the presence of an aberrantly spliced product from which exon 4 is excluded. Aberrant splicing could sufficiently account for the low level of functional tk transcript and thus the TK- phenotype in T2, although potential contributions from other mechanisms cannot be excluded.

Alternative Splicing↗
PubMed1992

Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells.

In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of X-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp X-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats.

Adenine Phosphoribosyltransferase↗
PubMed1990

Loss of heterozygosity in mammalian cell mutagenesis: molecular analysis of spontaneous mutations at the aprt locus in CHO cells.

Loss of heterozygosity at previously heterozygous loci may occur by one of several possible mechanisms and account for a large fraction of all mutations occurring at such loci. In order to investigate loss of heterozygosity events, we have chosen the aprt locus of Chinese hamster ovary (CHO) cells as our model since it is readily available in either heterozygous or hemizygous form. Cloning and sequencing of the two heterozygous aprt alleles from the CHO derivative D423 identified a single polymorphic site, which does not create a restriction fragment length polymorphism. In order to evaluate the loss of heterozygosity events at this locus, we devised a method that creates an artificial restriction fragment length polymorphism in one of these two alleles as a direct consequence of enzymatic amplification. Restriction enzyme digestion of the amplified sequences can then conveniently identify the genotype of the DNA sample. This same methodology also provides for the selective cloning of only one allele of a heterozygous pair into a plasmid vector for subsequent DNA sequence analysis, and can be easily adapted to other situations requiring the analysis of single base changes at a particular position within known sequences. Using this technique, we have determined that 16/37 (43%) spontaneous APRT- mutants had undergone a loss of heterozygosity event.

Adenine Phosphoribosyltransferase↗
PubMed1989

Perspectives on UV light mutagenesis: investigation of the CHO aprt gene carried on a retroviral shuttle vector.

The extent to which the cellular processing of shuttle vector-carried genes reflects that of endogenous chromosomal loci has been a subject of considerable controversy. In order to address this issue, we have developed a retroviral-based shuttle vector carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene stably integrated into the genome to be used for studying mutational specificity in mammalian cells. Initially, we have characterized a collection of UV-induced mutants in a CHO cell background. We have therefore been able to directly compare this shuttle vector data to that previously obtained for UV-induced mutation at the endogenous CHO (aprt) locus. Although some potential differences between the two spectra have been noted, there appears to be a remarkable similarity in the distribution and site specificity of UV-induced mutations. These similarities extend to extrachromosomal shuttle vectors as well and consolidate the role of shuttle vectors as powerful analytical tools for studying mechanisms of point mutagenesis in mammalian cells.

Adenine Phosphoribosyltransferase↗
PubMed1989

Mutational specificity studies of endogenous mammalian cell loci: methodological aspects.

We report here the details of a modified cloning method first described by Seed et al. (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells. The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage lambda vector. Recombinant phage form blue plaques on a non-suppressor, lacZ amber host. We have determined the sensitivity of the method. Homologous recombination frequency is in the order of 10(-3), 6-7 orders of magnitude above non-homologous events. Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector. A background of plaques is observed along with the desired blue plaques. Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events.

Adenine Phosphoribosyltransferase↗
PubMed1988

Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells.

The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage.

Adenine Phosphoribosyltransferase↗
PubMed1988

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

The spectrum of spontaneous mutation of an endogenous mammalian cell gene has been determined at the DNA sequence level. Thirty independent spontaneous APRT- mutations were cloned and subsequently completely sequenced. Twenty-seven contained single base substitutions. Of these, 22 were G.C to A.T transitions, suggesting a major role for the deamination of cytosine in spontaneous mutagenesis of Chinese hamster ovary cells. The remaining mutants included a tandem double substitution, a -1 frameshift, and a 17-base-pair deletion flanked by a 2-base-pair direct repeat. Many of the independently recovered mutants were clustered at sites of multiple occurrence (hot spots). One site accounted for greater than 25% of all independently recovered events. Mutations were generally located within the coding sequence, although two mutations occurred within the consensus sequence for a 3' splice site.

Adenine Phosphoribosyltransferase↗