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Biomedical subjects

D Schlessinger

Publications and source records attributed to D Schlessinger.

At least 91 records · Page 5Linked to original sources
PubMed1994

HPRT yeast artificial chromosome transfer into human cells by four methods and an involvement of homologous recombination.

The transfer of a yeast artificial chromosome (YAC) into mouse and human cell lines was effected by four methods, and the efficiency and integrity of the incorporated YAC DNA were compared. A 500 kb YAC containing the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was transferred more efficiently by polyethylene glycol-mediated fusion than by lipofection, electrofusion, or electroporation. Southern blot analysis demonstrated that PEG fusion lines yielded fragments of the size of the original YAC clone, whereas lipofection and electroporation did not. Two of 53 fusion lines showed 6-thioguanine resistance and confirmatory disruption of the HPRT gene in the YAC DNA, suggesting that the YAC DNA was integrated by homologous recombination with the endogenous HPRT gene region.

Animals↗
PubMed1994

Chimeric YACs were generated at unreduced rates in conditions that suppress coligation.

Chimerism is a major limitation of current YAC libraries. A method based on partially filled-in ends of restriction fragments was designed to avoid coligation as a possible source of chimeras. Model experiments using plasmid DNA as an insert showed that coligation was clearly avoided by this method. Pilot collections of YACs with an average insert size of 650kb were then constructed with and without the partial fill-in treatment. Starting from a mixture of a equal amounts of human and mouse DNA, none of 108 clones was positive by hybridization with both Alu and B2 probes, again suggesting that coligation was effectively blocked. However, 4 out of 10 clones still hybridized to 2 or more locations by FISH on chromosomes in human metaphase spreads, level similar to that in the clones made without the partial fill-in step. These results strongly suggest that chimeric clones generally arise by a mechanism independent of coligation, presumptively based on recombination.

Animals↗
PubMed1994

Structure of the human CCG1 gene: relationship between the exons/introns and functional domain/modules of the protein.

The human CCG1 gene, encoding CCG1/TAFII250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA. Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long, has been recovered in a YAC, which provides a route to the further study of module function.

Base Sequence↗
PubMed1994

Stability of YACs containing ribosomal or RCP/GCP locus DNA in wild-type S. cerevisiae and RAD mutant strains.

About 2% of human YAC clones, including tandemly repeated segments color vision pigment DNA, ribosomal DNA and alphoid DNA have been reported to be inherently unstable in yeast hosts, producing more stable deletion products. YACs containing color vision red pigment gene DNA or 1.5 rDNA tandem repeat units were transformed into hosts bearing lesions at the RAD1, RAD6, RAD51, or RAD52 loci. YACs susceptible to deletion during outgrowth of wild-type cells (or in preliminary experiments, in RAD6 transformants) were stable for up to 100 generations or more in the other strains. Thus both the RAD1 and RAD51/RAD52 epistatic pathways are apparently involved in the instability of YACs containing tandem repeat loci, presumably during recombination-based deletion formation; and a yeast host disarmed in these pathways will likely maintain YACs intact that are otherwise unstable.

Animals↗
PubMed1994

BLOCK-based PCR markers to find gene family members in human and comparative genome analysis.

Degenerate primer pairs that include consensus sequences of evolutionary conserved portions of protein families (BLOCKs or ancient conserved regions) can be used to screen by polymerase chain reaction (PCR) for cognate cDNAs and YACs through much of phylogeny. Nine such primer pairs were developed, and five with sites on human chromosomes 7 or X were shown to identify YACs from chromosome-specific locations, including a candidate for a new zinc finger gene in Xq28. When linked to contig-based genomic maps, such BLOCK-based PCR assays may provide a route to recover the members and study the development of families containing up to 40% of genes, in genomes as diverse as humans, nematodes, and yeast.

Actins↗
PubMed1994

Increased retention of small chromosomes in a novel yeast mutant.

MSC201 and MSC202 are novel yeast mutant strains in which a 10-kb linear yeast artificial chromosome (YAC) was stably maintained in unselective medium during mitotic cell division. After culturing for 35 cycles of cell division, about 50% of MSC cells retain YAC3 DNA, compared to 0.001% of the wild-type AB1380 strain. Southern blot hybridization analysis with pBR322 DNA as the probe showed that in the MSC transformants, YAC DNA remained in a linear form free of cellular chromosomal DNA. The msc201 mutation was shown to be recessive by the rapid loss of the YAC in a diploid strain made by mating with AB1375, which has a genetic background similar to that of the MSC strain. Linear YAC DNA with a centromere was stabilized in MSC201 better than a linear construct lacking a centromere sequence.

Chromosomes, Artificial, Yeast↗
PubMed1993

Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes.

Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids we mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene.

Base Sequence↗
PubMed1993

Alu and L1 sequence distributions in Xq24-q28 and their comparative utility in YAC contig assembly and verification.

The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant, and using software based on that of M. V. Olson et al. (1986, Proc. Natl. Acad. Sci. USA 83: 7826) to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes.

Chromosome Mapping↗
PubMed1993

1.5-Mb YAC contig in Xq28 formatted with sequence-tagged sites and including a region unstable in the clones.

A contig of 20 yeast artificial clones (YACs) has been assembled across 1.5 Mb of Xq28 and formatted with nine previously reported probes and nine STSs developed from the sequence of probes and end fragments of YACs. YAC end fragments were obtained by subcloning, Alu-vector PCR, or primer-ligation PCR methods. Eighteen of the YACs were recovered from a library specific for Xq24-q28; two that fill a gap were obtained from a second library made from total human DNA. One region, containing probes pX78c and 2A1.1, was unstable in YACs, but it was possible to generate a self-consistent map of DNA over the entire contig. Overlaps were confirmed by Southern blot analyses of YAC DNAs, and pulsed-field gel electrophoresis confirmed the extent of the contig and identified at least four CpG islands in the region.

Base Sequence↗
PubMed1993

High-resolution mapping of probes near the X-linked lymphoproliferative disease (XLP) locus.

Fluorescence in situ hybridization (FISH) was employed in high-resolution mapping of probes near the X-linked lymphoproliferative disease (XLP) locus. The map includes the DXS42, DXS12, DXS6, DXS982, DXS739, DXS75, DXS100, DXS10, and DXS177 loci. Metaphase analysis showed that DXS12 and DXS42 mapped to proximal Xq25, while DXS10 and DXS177 mapped to proximal Xq26.1. DXS6, DXS982, DXS739, DXS75, and DXS100 were in Xq25. The order of probes deduced from interphase FISH was: Xq24-(DXS12, DXS42)-DXS6-DXS982-DXS739-DXS75-DXS100+ ++-DXS10-DXS177-Xq26.2. We estimate that the entire region between DXS12 and DXS177 is about 7 Mb. Our previous study indicated that all three XLP deletions (63-3, 66-1, and 43-4) lacked DXS739. We now report that DXS75 and DXS982 are also missing in these deletions. Using interphase FISH measurements, we estimate that 2 Mb are absent in 63-3, and 4 Mb are absent in 66-1 and 43-4. This FISH map confines the XLP candidate gene region to a 2-Mb interval between DXS6 and DXS100 and places DXS100 distal to the XLP locus. This study also demonstrates that small probes (0.6 to 3.6 kb) can be utilized in FISH.

Chromosome Deletion↗
PubMed1993

Isochores and CpG islands in YAC contigs in human Xq26.1-qter.

GC levels were assessed at 37 loci across 30 Mb of Xq26.1-qter, a region physically mapped in overlapping yeast artificial chromosome clones. In 8 Mb of R band Xq26, GC is relatively high (up to 44%) in the proximal 4 Mb and relatively low (40-41%) in the distal 4 Mb. Consistently low GC values (38-41%) are observed in G band Xq27. In contrast, further toward the telomere in Xq28, the GC level rises progressively to reach 52% at 2 to 4 Mb from the end of the chromosome; this region is delimited by low GC loci. Across these regions of Xq, the content of rare-cutter restriction enzyme sites containing CpG, including "CpG islands" in the most completely mapped Xq26-27.1 region, is correlated with GC level. Isochore mapping can thus provide one index of putative gene content across mapped regions.

Chromosome Banding↗
PubMed1993

Ordered shotgun sequencing, a strategy for integrated mapping and sequencing of YAC clones.

Ordered shotgun sequencing proposes to organize the mapping and sequencing of YACs with a hierarchical strategy that incorporates a feedback loop. Building on current protocols, a YAC is subcloned into plasmids, plasmid insert ends are sequenced, and the sequences are overlapped to create a partial map. Complete sequencing then starts with plasmids whose end-sequence tracts have overlapped, but to a minimal extent. The next plasmids to be sequenced are again selected for least overlap, striking out progressively to span the YAC with minimal directed gap-filling. Simulations support its feasibility and indicate that during the generation of the complete sequence, the approach facilitates the early choice of regions for selective sequencing, for example, for coding units. The sequencing of plasmids would also require less redundancy, and discriminate repetitive sequences more easily, than random sequencing across larger clones. The overall effort scales with YAC size and can be further reduced by additional mapping information.

Chromosome Mapping↗
PubMed1993

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes.

For determination of the extent to which ribosomal DNA (rDNA) is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.

Animals↗
PubMed1993

Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes.

Colored chromosome staining patterns, termed chromosomal 'bar codes' (CBCs), were obtained on human chromosomes by fluorescence in situ hybridization (FISH) with pools of Alu-PCR products from YAC clones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, individual color and relative signal intensity of each 'bar' could be modified depending on probe selection and labeling procedures. Alu-PCR amplification products were generated from 31 YAC clones which mapped to 37 different chromosome bands. For multiple color FISH, Alu-PCR amplification products from various clones were either biotinylated or labeled with digoxigenin. Probes from up to twenty YAC clones were used simultaneously to produce CBCs on selected human chromosomes. Evaluation using a cooled CCD camera and digital image analysis confirmed the high reproducibility of the bars from one metaphase spread to another. Combinatorial FISH with mixtures of whole chromosomes paint probes was applied to paint seven chromosomes simultaneously in different colors along with a set of YAC clones which map to these chromosomes. We discuss the potential to construct analytical chromosomal bar codes adapted to particular needs of cytogenetic investigations and automated image analysis.

Chromosome Banding↗