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D Schlessinger

Publications and source records attributed to D Schlessinger.

At least 127 records · Page 7Linked to original sources
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

Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome.

The sequence of 20,114 bp of DNA including the human glucose-6-phosphate dehydrogenase (G6PD) gene was determined. The region included a prominent CpG island, starting about 680 nucleotides upstream of the transcription start site, extending about 1050 nucleotides downstream of the start site, and ending just at the start of the first intron. The transcribed region from the start site to the poly(A) addition site covers 15,860 bp. The sequence of the 13 exons agreed with published cDNA sequence and for the 11 exons tested, with the corresponding sequence in a yeast artificial chromosome (YAC). The latter confirms YAC cloning fidelity at the DNA sequence level. Sixteen Alu sequences constitute 24% of the total sequence tract. Four were outside the borders of the mRNA transcript of the gene; all the others were found in a large (9858 bp) intron between exons 2 and 3. Two Alu clusters each contain Alus lying between the monomers of another.

Amino Acid Sequence↗
PubMed1991

In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA.

From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to +/- 0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7-10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions.

Base Sequence↗
PubMed1990

Yeast artificial chromosomes: tools for mapping and analysis of complex genomes.

Libraries of yeast artificial chromosomes (YACs) are representative of complex genomes, and typical YACs contain single fragments of DNA that are up to a megabase or more in size, are stable during growth, and are faithful to genomic DNA. Such YACs may permit (1) the use of complete gene units for a number of research and medical purposes; and (2) the assembly of chromosome-sized contigs in a single map that unifies genetic and physical data.

Animals↗
PubMed1990

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences.

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.

Animals↗
PubMed1990

Vanadate promotes reactivation and iontophoresis-induced ocular shedding of latent HSV-1 W in different host animals.

Vanadate is a potent inhibitor of calcium stimulated ATPase, Na, K-ATPase, and may have adrenergic activity. Using the iontophoresis method, we compared vanadate to a BSS control and the standard iontophoresis model (6-hydroxydopamine/epinephrine) by measuring induced ocular shedding of latent HSV-1 in different host animals. Latent trigeminal ganglionic infections were established in Balb/c mice and New Zealand rabbits following corneal inoculation with HSV-1 [W] strain, and later confirmed by cocultivation. Latently-infected animals (greater than 1 month post-infection) were divided into three treatment groups. Each group was iontophoresed with BSS, vanadate 1% or 6-HD 1%, and then treated topically for 10 days with BSS, vanadate or epinephrine respectively. Reactivation and recovery of latent HSV-1 was detected by daily ocular swabbing, plating, and observing progressive viral growth in Vero cells. The vanadate group had more virus-positive eyes than the BSS control group in mice, (8/32 vs. 1/32 P less than .01), and also in rabbits (14/20 vs 6/22 P less than .01). Virus-positive animals and total positive swabs were also higher for vanadate than BSS in both mice and rabbits. Furthermore, while vanadate was associated with fewer virus-positive eyes than 6-HD & EPI (8/32 vs. 17/32 P less than .02) in mice, there were no significant differences in rabbits. We conclude that vanadate promotes ocular shedding of latent HSV-1, and may act through an adrenergic mechanism.

Animals↗
PubMed1990

Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes.

One hundred twenty-seven yeast strains with artificial chromosomes containing Xq24-Xqter human DNA were obtained starting from a human/hamster somatic cell hybrid. The clones were characterized with respect to their insert size, stability, and representation of a set of Xq24-Xqter DNA probes. The inserts of the clones add up to 19.3 megabase (Mb) content, or about 0.4 genomic equivalents of that portion of the X chromosome, with a range of 40-650 kb in individual YACs. Eleven clones contained more than one YAC, the additional ones usually having hamster DNA inserts; the individual YACs could be separated by extracting the total DNA from such strains and using it to retransform yeast cells. One of the YACs, containing the probe for the DXS49 locus, was grossly unstable, throwing off smaller versions of an initial 300-kb YAC during subculture; the other YACs appeared to breed true on subculture. Of 52 probes tested, 12 found cognate YACs; the YACs included one with the glucose-6-phosphate dehydrogense gene and another containing four anonymous probe sequences (DX13, St14, cpx67, and cpx6). Xq location of YACs is being verified by in situ hybridization to metaphase chromosomes, and fingerprinting and hybridization methods are being used to detect YACs that overlap.

Animals↗
PubMed1989

Human ribosomal DNA: conserved sequence elements in a 4.3-kb region downstream from the transcription unit.

The sequence of 4366 bp of nontranscribed spacer (NTS) human ribosomal DNA (rDNA) located downstream from the 3' end of the transcription unit has been determined. The NTS rDNA is rich in pyrimidine nucleotides (31% T and 30% C) that tend to occur on the coding strand in runs of simple sequence repeats. Other highly repetitive sequence elements are also represented, including tracts of (dA-dC)26 and (dG-dT)29 on the coding strand downstream from the putative termination of transcription. Still farther downstream, two Alu repeat sequences are found. Such sequences are also found in rat DNA at comparable locations, consistent with the possibility of a comparable functional role.

Base Composition↗
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

Processing pathway of Escherichia coli 16S precursor rRNA.

Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA.

Base Sequence↗
PubMed1989

Escherichia coli 16S rRNA 3'-end formation requires a distal transfer RNA sequence at a proper distance.

The 16S rRNA species in bacterial precursor rRNAs is followed by two evolutionarily conserved features: (i) a double-stranded stem formed by complementary sequences adjacent to the 5' and 3' ends of the 16S rRNA; and (ii) a 3'-transfer RNA sequence. To assess the possible role of these features, plasmid constructs with precursor-specific features deleted were tested for their capacity to form mature rRNA. Stem-forming sequences were dispensable for both 5' and 3' terminus formation; whereas an intact spacer tRNA positioned greater than 24 nucleotides downstream of the 16S RNA sequence was required for correct 3'-end maturation. These results suggest that spacer tRNA at an appropriate location helps form a conformation obligate for pre-rRNA processing, perhaps by binding to a nascent binding site in preribosomes. Thus, spacer tRNAs may be an obligate participant in ribosome formation.

Base Sequence↗
PubMed1989

Complete human rDNA repeat units isolated in yeast artificial chromosomes.

Human ribosomal DNA has been inferred to be organized in tandem repeat units of 44 kb, of which only 13 kb is transcribed into preribosomal RNA. Unfortunately, it has remained difficult to examine the intact repeat structure directly, because even a single repeat unit is too large to be accommodated in conventional cloning systems. Here we report the isolation of intact repeat units using yeast artificial chromosomes as a cloning tool. With a spacer sequence specific to human ribosomal DNA used as a probe, 27 clones were identified among 17,000 YACs (about 0.7 genomic equivalent of total human DNA). Fourteen clones contained only a small portion of rDNA; the other 13 contained most or all of the rDNA repeat unit, and 8 of those were studied in further detail. They contained 1 to 1.5 repeat units of rDNA with all of the expected EcoRI and HindIII fragments. These clones provide possible starting material for the analysis of expression of a single unit of rDNA. Unexpectedly, however, only the four smaller clones (70 to 90 kb) were completely composed of standard rDNA sequences; four larger clones (up to 950 kb in length) contained additional "non-rDNA" sequences, at either one or both ends of the repeat unit. Analysis of these atypical rDNA clones suggests that their inserts either are scattered in the genome or are localized in a nucleolar organizer region that is more complex than previously recognized.

Base Sequence↗
PubMed1989

Yeast artificial chromosomes with 200- to 800-kilobase inserts of human DNA containing HLA, V kappa, 5S, and Xq24-Xq28 sequences.

Sequences hybridizing to several human gene probes have been recovered as cloned inserts in yeast artificial chromosomes (YACs). Among 2300 YACs made from human leukocyte DNA (totaling about 0.1 genomic equivalent of human DNA) we have found two, 200 and 780 kilobases (kb), containing sequences of V kappa I immunoglobulin (V = variable); one, 240 kb, with class I HLA; and 11, 200-800 kb, with 5S rRNA-encoding DNA (rDNA). Fifty human YACs from a hamster-human cell hybrid with only the Xq24-Xq28 portion of the X chromosome include one that contains two anonymous probe sequences, DX13 and St14, previously inferred by indirect means to lie within about 70 kb of one another in Xq28. The YACs specific for human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human-specific. Furthermore, the human YACs, formed in a 280-fold excess of hamster DNA, do not hybridize to a hamster DNA probe, indicating that individual YACs do not contain a combination of human and hamster DNA. To confirm that sequences are not scrambled, the YACs containing V kappa I or DX13 and St14 sequences were shown to produce restriction fragments identical in mobility to fragments detected by the same probes in total human DNA digested with the same enzymes. YACs may therefore provide large clones to bridge gene mapping at the chromosome level to molecular analyses of small fragments of genomic DNA.

Animals↗
PubMed1989

Transcription and processing of RNA from mouse ribosomal DNA transfected into hamster cells.

Transcription of mouse genes coding for rRNA in CHO cells was promoter dependent at levels 3 to 10% of that of endogenous rRNA synthesis. Northern (RNA) and S1 nuclease mapping analyses demonstrated that transcription proceeds through the entire gene segment coding for rRNA in transfected constructs and continues, at least in some cases, into the adjoining plasmid sequences. S1 nuclease mapping also detected some processing cleavages in the transcripts, including those at the 3' terminus of 18S rRNA, those at the rapidly cleaved site at +650 in the external transcribed spacer, and those at a previously uncharacterized, rapidly cleaved site in the internal transcribed spacer. Deletion of sequences upstream or downstream from the promoter generally had no measurable effect on the level of transcription, but deletion of a 300-base-pair XhoI-XhoI fragment starting 1,287 base pairs from the transcription start site sharply increased the steady-state level of rRNA. Effects on processing were harder to test, because many intermediates are too unstable to detect even by S1 nuclease mapping; however, the data suggest that RNAs with deletions in the external transcribed spacer are processed poorly at distal sites. Processing at some sites may thus depend on interactions involving distant segments of rRNA.

Animals↗
PubMed1988

Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.

Animals↗