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D M Kurnit

Publications and source records attributed to D M Kurnit.

At least 55 records · Page 3Linked to original sources
PubMed1991

Analysis of proteins synthesized by 9.5 day mouse embryos: determination of cardiac and noncardiac proteins.

To catalog polypeptides that were specific to developing hearts, we separated 35S-methionine-labeled 9.5 day mouse embryos into cardiac and noncardiac (carcass) components. Two-dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or increased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac-specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac-specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac-specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two-dimensional gels.

Animals↗
PubMed1991

DNA sequences surrounding the centromere of chromosome 21.

The mapping and sequencing of two clones that surround the centromere of chromosome 21 are presented. These clones specify the most proximal known low-order repeat on 21p (p21-7D) and the most proximal known single-copy sequence on 21q (pUT-B37 at locus D21S120).

Base Sequence↗
PubMed1990

Recombination-based screening for genes on chromosome 21.

In previous work, a reasonably large number of genes was found to be on chromosome 21. This frequency demands the creation of new techniques to investigate the transcription of this small human autosome, and to relate these findings to the phenotype of Down syndrome. Here we describe the elaboration of new vectors and hosts, which in conjunction with flow-sorted cosmid libraries of chromosome 21 and genic (cDNA) libraries of relevant human tissues, will permit us to examine transcription of chromosome 21.

Chromosome Mapping↗
PubMed1990

Nondisjunction of chromosome 21.

Chromosome heteromorphisms and restriction fragment length polymorphisms were used to study the origin of the extra chromosome in 54 trisomy 21 conceptuses. The parental origin was determined in 43 cases, with 39 (91%) being maternally and 4 (9%) parentally derived. Analysis of recombination demonstrated the presence of one or two cross-overs in most cases for which sufficient information was available, suggesting that failure to pair/exchange at meiosis I is relatively unimportant in the genesis of trisomy 21.

Adult↗
PubMed1990

The murine situs inversus viscerum (iv) gene responsible for visceral asymmetry is linked tightly to the Igh-C cluster on chromosome 12.

The iv gene controls left-right determination during murine organogenesis. To map this gene, we analyzed backcross progeny produced by mating (C57BL/6J X MEV/Ty)F1-iv/+heterozygotes to C57BL/6J-iv homozygotes. Hybridization of a murine ecotropic virus probe and several homeotic box gene probes coupled with analysis of dominant visible markers enabled us to exclude the iv locus from much of the mouse genome. Spurred by a recent report that mapped the iv gene to mouse chromosome 12 which was not excluded by our previous work, we used the polymerase chain reaction on our larger cohort to determine that the iv gene is indeed linked tightly to the Igh-C locus on this chromosome: we observed 0/156 recombinants between the iv and Igh-C loci. Combining data from the two studies demonstrates that the murine iv gene is close (1/201 recombinants) to the Igh-C cluster on chromosome 12.

Animals↗
PubMed1990

Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo.

Three major difficulties have hindered the general application of in vivo recombination techniques to library screening: (i) the original selection could not be applied to libraries prepared in phage vectors lacking amber mutations, (ii) nonirradiated packaging extracts gave high backgrounds even when amber mutated vectors were used, and (iii) most red- vectors lacked rap, a recently discovered phage gene promoting phage-plasmid recombination. Here we describe a selection scheme for phage bearing suppressor tRNA plasmids, which relies upon an Escherichia coli host bearing an amber mutation in the dnaB gene. The selection is tight enough to allow library screening by recombination, is applicable to almost every phage vector in common use, and overcomes the background associated with nonirradiated packaging extracts. We also describe an ancillary plasmid that supplies the rap gene function in trans, permitting the recombination level to be raised fruitfully in phage libraries lacking endogenous rap. Finally, we demonstrate simple procedures for preparing and detecting phages that have lost integrated suppressor tRNA plasmids by homologous recombination.

Base Sequence↗
PubMed1989

The P2 phage old gene: sequence, transcription and translational control.

The old (overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage lambda growth [Sironi et al., Virology 46 (1971) 387-396; Lindahl et al., Proc. Natl. Acad. Sci. USA 66 (1970) 587-594]. Specialized transducing lambda phages, which lack the recombination region, can be selected by plating lambda stocks on E. coli that carry the old gene on a prophage or plasmid [Finkel et al., Gene 46 (1986) 65-69]. Deletion and sequence analyses indicate that the old-encoded protein has an Mr of 65,373 and that its transcription is leftward. Primer extension analyses locate the transcription start point near the right end of the virion DNA. A bacterial mutant, named pin3 and able to suppress the effects of the old gene, has been isolated [Ghisotti et al., J. Virol. 48 (1983) 616-626]. In a pin3 mutant strain, carrying the old gene on a prophage or plasmid, the amount of old transcript is greatly reduced. The effect of the pin3 mutation is abolished by the wild-type allele of argU, an arginine tRNA that reads the rare Arg codons AGA and AGG, which are used for eight of the 14 Arg codons in the old gene. Thus the pin3 allele probably stalls translation of the old mRNA, causing this mRNA to be degraded. Isoelectric focusing and electrophoretic analysis identify the old gene product as a basic protein of approx. 65 kDa.

Amino Acid Sequence↗
PubMed1989

Sumo15A: a lambda phasmid that permits easy selection for and against cloned inserts.

We report the construction of a phasmid vector, Sumo15A, designed for recombination-based screening of recombinant DNA libraries [Seed, Nucleic Acids Res. 11 (1983) 2427-2445]. This vector permits rapid selection in Escherichia coli for homology-mediated integration and excision between homologous DNA inserts cloned in a supF-carrying plasmid and in Sumo15A. The region available for recombination spans the homologous sequence shared by the plasmid and the phasmid. SupF is the selection tool that we used. Efficient selection for supF expression by Sumo15A requires recombination mediated by the lambda phage red gene, which promotes homologous recombination between phage and plasmid DNAs. Counterselection against supF expression by Sumo15A occurs because the presence of a pSC101-derived plasmid replicon in this phasmid permits the growth of Sumo15A as a plasmid in a specialized host, E. coli strain DK37. In strain DK37, Sumo15A cannot replicate as a phage, and the presence of a plasmid-carrying supF is lethal to cells plated on galactose plates. This scheme was developed to select for sequences that are transcribed from chromosomes of interest.

Bacteriophage lambda↗
PubMed1989

Characterization of an unusual and complex chromosome 21 rearrangement using somatic cell genetics and cloned DNA probes.

In a previous case of a newborn infant with typical Down syndrome, chromosome analysis indicated the presence of an unusual and complex translocation of chromosome 21. The patient's cells contained one normal chromosome 21 and a rearranged, F group-sized submetacentric chromosome. This abnormal chromosome appeared to involve duplication of the distal portion of 21q with translocation to the short arm, and a deletion of C-band-positive centromeric heterochromatin. Using linearly ordered cloned DNA probes, we report the detailed molecular examination of this abnormal chromosome, which has been isolated on a hamster background in a hybrid cell line. Both short arm and pericentromeric sequences are present on this chromosome, as well as distal 21q sequences. However, a substantial portion of proximal 21q is deleted. The distal boundary of this deleted section can be pinpointed within the region between two loci (D21S8 and D21S54), a distance of about 5,000 kb. This study illustrates the power of using precisely mapped, linearly ordered DNA probes to characterize this type of rearrangement. In addition, this hybrid cell line can also be used as a member of a mapping panel to map DNA sequences regionally on chromosome 21.

Animals↗
PubMed1988

Isolation of DNA sequences on human chromosome 21 by application of a recombination-based assay to DNA from flow-sorted chromosomes.

By merging two efficient technologies, bivariate flow sorting of human metaphase chromosomes and a recombination-based assay for sequence complexity, we isolated 28 cloned DNA segments homologous to loci on human chromosome 21. Subregional mapping of these DNA segments with a somatic cell hybrid panel showed that 26 of the 28 cloned DNA sequences are distributed along the long arm of chromosome 21, while the other 2 hybridize with sequences on the short arm of both chromosome 21 and other chromosomes. This new collection of probes homologous to chromosome 21 should facilitate molecular analyses of trisomy 21 by providing DNA probes for the linkage map of chromosome 21, for studies of nondisjunction, for chromosome walking in clinically relevant subregions of chromosome 21, and for the isolation of genes on chromosome 21 following the screening of cDNA libraries.

Animals↗
PubMed1988

Trisomy 21 (Down syndrome): studying nondisjunction and meiotic recombination by using cytogenetic and molecular polymorphisms that span chromosome 21.

By combining molecular and cytogenetic techniques, we demonstrated the feasibility and desirability of a comprehensive approach to analysis of nondisjunction for chromosome 21. We analyzed the parental origin and stage of meiotic errors resulting in trisomy 21 in each of five families by successfully using cytogenetic heteromorphisms and DNA polymorphisms. The 16 DNA fragments used to detect polymorphisms spanned the length of the long arm and detected recombinational events on nondisjoined chromosomes in both maternal meiosis I and maternal meiosis II errors. The meiotic stage at which errors occurred was determined by sandwiching the centromere between cytogenetic heteromorphisms on 21p and an informative haplotype constructed using two polymorphic DNA probes that map to 21q just below the centromere. This study illustrates the necessity of combining cytogenetic polymorphisms on 21p with DNA polymorphisms spanning 21q to determine (1) the source and stage of meiotic errors that lead to trisomy 21 and (2) whether an association exists between nondisjunction and meiotic recombination.

Chromosome Banding↗