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M V Olson

Publications and source records attributed to M V Olson.

At least 19 recordsLinked to original sources
PubMed1992

Metaphase and interphase cytogenetics with Alu-PCR-amplified yeast artificial chromosome clones containing the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2.

A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.

Base Sequence↗
PubMed1992

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.

Base Sequence↗
PubMed1991

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.

Acyltransferases↗
PubMed1991

Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes.

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.

Animals↗
PubMed1991

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.

Animals↗
PubMed1991

The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion.

A 680-kb yeast artificial chromosome (YAC) that contains a functional copy of the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3' end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.

Animals↗
PubMed1991

Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

Blotting, Southern↗
PubMed1990

Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping.

A general strategy for cloning and mapping large regions of human DNA with yeast artificial chromosomes (YAC's) is described. It relies on the use of the polymerase chain reaction to detect DNA landmarks called sequence-tagged sites (STS's) within YAC clones. The method was applied to the region of human chromosome 7 containing the cystic fibrosis (CF) gene. Thirty YAC clones from this region were analyzed, and a contig map that spans more than 1,500,000 base pairs was assembled. Individual YAC's as large as 790 kilobase pairs and containing the entire CF gene were constructed in vivo by meiotic recombination in yeast between pairs of overlapping YAC's.

Base Sequence↗
PubMed1990

Spatial normalization of one-dimensional electrophoretic gel images.

A strategy for using processed, digitized images of one-dimensional electrophoretic gels to facilitate the analysis of large sets of overlapping clones is described. The images are acquired from fluorescently stained gels or from transilluminated gel photographs using a cooled, solid-state charge-coupled device camera. By employing sets of bands in the size-standard lanes as reference points, all the gel images are spatially normalized to a common reference template. After normalization, lane images from different gels can be compared as though the gels had been electrophoresed under identical, uniform-field conditions. Applications of this procedure to the analysis of a large set of overlapping lambda clones from chromosome VII of Saccharomyces cerevisiae and to the estimation of fragment sizes are illustrated.

Analog-Digital Conversion↗
PubMed1990

Second-generation approach to the construction of yeast artificial-chromosome libraries.

We describe an improved method for construction of yeast artificial-chromosome (YAC) libraries that contain large inserts of foreign DNA. The procedure consists of seven steps: (i) preparation of human DNA in agarose beads; (ii) partial digestion of the DNA with EcoRI; (iii) electrophoretic elimination of the smaller partial-digest fragments; (iv) ligation of the EcoRI fragments with vector arms in molten agarose; (v) hydrolysis of agarose with agarase; (vi) fractionation of the recombinant molecules by sucrose-gradient centrifugation; and (vii) transformation of yeast. More than 7000 colonies were obtained starting with 15 micrograms of human DNA, which was fractionated on a single sucrose gradient. The average size of these YACs was approximately 380 kb. It is estimated that the total length of human DNA present in the clones corresponds to 80% of the length of the human haploid genome. The results of screening the clones for a number of single-copy genes indicate that the clones reflect a nearly random sampling of the human genome. The efficiency of the cloning is sufficient to support the construction of multihit libraries for the human genome or for the genomes of other higher organisms.

Base Sequence↗
PubMed1990

Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction.

We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determine the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.

Base Sequence↗
PubMed1989

Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

Chromosomes, Fungal↗
PubMed1989

Separation of large DNA molecules by pulsed-field gel electrophoresis. A review of the basic phenomenology.

Pulsed-field gel electrophoresis is a method of separating large DNA molecules. The distinctive feature of this method is that the direction of the electric field is changed periodically. During the five years since Schwartz and Cantor introduced this technique, there has been dramatic progress in pulsed-field instrumentation and in associated electrophoretic methods. Progress has been driven by practical experience with little guidance from theory. In this review, the basic phenomenology of pulsed-field gel electrophoresis is summarized and some speculations are advanced about possible molecular mechanisms.

DNA↗
PubMed1989

Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.

Base Sequence↗
PubMed1988

Nonsense mutations in essential genes of Saccharomyces cerevisiae.

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.

Genes, Fungal↗