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J C Fuscoe

Publications and source records attributed to J C Fuscoe.

At least 19 recordsLinked to original sources
PubMed1992

Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions.

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) 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. In addition, the DNA sequence of the insertion mutation was determined. The insertion (229 bp) is coupled with a deletion (31 bp). An imperfect inverted repeat with flanking hprt DNA was identified and may be involved in the insertion event.

Animals↗
PubMed1992

Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.

Adult↗
PubMed1992

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.

Adult↗
PubMed1991

V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.

Base Sequence↗
PubMed1991

Workshop overview: new molecular techniques in genome analysis.

An overview of the application of various molecular techniques to the analysis of genomic DNA is presented. For the analysis of small-scale changes, the polymerase chain reaction (PCR), colony probe hybridization, mismatch hybridization, and denaturing gradient gel electrophoresis (DGGE) are providing information on mutations within prokaryotic and eukaryotic genes. For large-scale changes, fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), Southern blotting, multiplex PCR, hybridization of linked probes, and restriction enzyme mapping are permitting analysis of genomic alterations that are larger than point mutations but below the resolution of standard cytogenetic analysis. Many of these techniques, either alone or in combination, produce DNA that can be subjected to DNA sequence analysis, which provides the most detailed information regarding genomic changes.

Chromosome Aberrations↗
PubMed1991

Direct DNA sequencing of PCR products.

Experiments are described which elucidate some of the technical problems associated with the direct sequencing of polymerase chain reaction (PCR) amplified DNA. Sequencing primer purity, labeling methodology, and template preparation were explored. Conditions are presented for the routine sequencing of single- and double-stranded PCR products.

Base Sequence↗
PubMed1991

The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen.

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.

Animals↗
PubMed1989

An efficient method for selecting unique-sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in situ hybridization.

This paper describes an efficient procedure for selecting large numbers of unique-sequence or very low repeat-sequence probes from recombinant phage libraries. Probes were selected from the Charon 21A library LL21NS02 (made from DNA from human chromosome 21) in a multistep process in which (1) inserts from LL21NS02 were subcloned into Bluescribe plasmids, (2) plasmids were grown at high density in colonies on nitrocellulose, and (3) plasmids were selected as containing unique-sequence inserts if DNA from the colonies failed to hybridize, at low stringency, to radiolabeled total human DNA. In this manner, 1530 colonies were picked to form the library pBS-U21/1530. About 80% of the recombinants constituting pBS-U21/1530 were shown by Southern analysis to carry inserts that are present in only one copy in haploid genomic human DNA. Approximately 70% of the sequences mapped to human chromosome 21. Fluorescence in situ hybridization with DNA from pBS-U21/1530 allowed specific, intense staining of the number 21 chromosomes in metaphase spreads made from human lymphocytes.

Bacteriophage lambda↗
PubMed1989

Human chromosome-specific DNA libraries: construction and purity analysis.

We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.

Animals↗
PubMed1988

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium.

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.

Aflatoxin B1↗
PubMed1987

High-speed chromosome sorting.

Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

Animals↗
PubMed1987

Human chromosome-specific DNA libraries: use of an oligodeoxynucleotide probe to detect non-recombinants.

The frequencies of non-recombinants are directly determined in 21 human DNA libraries prepared from flow-purified chromosomes. A radiolabeled 18-base oligodeoxynucleotide, whose sequence spans the cloning site, stably hybridizes to non-recombinants. When the cloning site is interrupted by an insert, less stable hybridization occurs. Non-recombinant frequencies range from 1-34% with the frequency of false positives being less than 1%. Libraries are being prepared for each human chromosome as part of the National Laboratory Gene Library Project and the specifications of seven new libraries are presented.

Base Sequence↗
PubMed1987

Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.

Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.

Animals↗
PubMed1986

DNA repair genes of mammalian cells.

In the Chinese hamster ovary (CHO) cell line, various mutations affecting DNA repair have been obtained. Mutants that belong to 5 genetic complementation groups for ultraviolet (UV) sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum (XP) were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutations analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has a very high frequency of sister chromatid exchange (SCE), are both corrected by chromosome 19. Efforts are underway to isolate complementing repair genes by DNA-mediated gene transfer. The human gene that corrects mutant EM9 and the hamster gene that corrects UV135 (UV Group 5) have been introduced by cotransfer of genomic DNA and the dominant selectable marker gpt (guanine phosphoribosyltransferase) gene. In each case, the DNA repair function was co-selected based on resistance to 5-chlorodeoxyuridine (CldUrd) or repeated UV irradiation, respectively. The presence of a functional human repair gene in the EM9 transformants is shown by the presence of common human DNA sequences on some fragments produced by restriction enzyme cleavage. In UV135, transfer of a repair gene is indicated by a colony distribution containing "jackpots" and by instability of the resistant phenotype.

Animals↗
PubMed1986

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells.

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.

Animals↗
PubMed1986

Construction of fifteen human chromosome-specific DNA libraries from flow-purified chromosomes.

We report the construction of 15 human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow sorting and the DNA was extracted and cleaved with HindIII before cloning into the lambda vector Charon 21A. A sensitive miniblot hybridization method was used to monitor the physical and biochemical steps in the cloning procedure. Using this method, we have developed a highly efficient protocol for producing large numbers of recombinant phage from 0.2-1.0 X 10(6) sorted chromosomes. DNA from the following chromosomes was cloned: #4, 6, 8, 9, 11, 13, 14 + 15, 16, 17, 18, 19, 20, 21, 22 and Y. These libraries are available to the scientific research community and will be valuable in the genetic analysis of the human genome.

Cell Line↗
PubMed1986

Molecular characterization of the purity of seven human chromosome-specific DNA libraries.

We have characterized at the molecular level seven chromosome-specific libraries constructed in phage lambda Charon 21A from flow-sorted human chromosomes. The purity of libraries prepared from chromosomes sorted from hamster X human cells was estimated by species-specific hybridization and ranged from 48% to 83% of clones containing human inserts. Among libraries of chromosomes from human cells, mass screenings were made for repetitive sequences and 20 clones from the #18 and #20 libraries were analyzed in detail. Ten to fifteen percent of all clones contain sequences which can be mapped; 80-100% of these derive from the intended chromosome of origin, demonstrating very high purity and a 35 X enrichment of chromosome-specific sequences over a total genomic library. The two libraries contain a high, though dissimilar, percent of repeat-containing clones; the #18 library has 55% repetitive clones and the #20 library 85%. This dissimilarity may be due to a difference in insert size distribution, since the #18 library has smaller inserts than the #20. This could be caused by variation in extent of digestion of insert DNA and/or differences in sequence organization between the two chromosomes. A method more sensitive than conventional plaque-lift screening was used to detect repetitive inserts; in this way nearly all repetitive clones could be eliminated before purification of their DNAs.

Animals↗