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D M Kurnit

Publications and source records attributed to D M Kurnit.

At least 37 records · Page 2Linked to original sources
PubMed1996

Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene.

To study gene regulation mediated by chromatin in mammals, we isolated the human (SUPT4H) and murine (Supt4h) counterparts of the yeast gene encoding SPT4; the product of this gene presumably interacts with the products of the mammalian homologues (which we have also cloned) of yeast SPT5 and SPT6, thereby modulating chromatin formation and activity. We isolated two different sized human SUPT4H cDNA clones (1464 and 728 nt) and one murine Supt4h (688 nt) cDNA clone; all three encode the same 117-amino-acid protein with conservation of the zinc finger motif found in SPT4. Conservation of this zinc finger motif from yeast to mouse and human implies functional importance. Although the overall sequence homology at the DNA level between the human 728-nt transcript and the murine 688-nt transcript is only 78.4%, the DNA sequence homology is 97.7% within the coding region. At the protein level, the amino acid sequences of the translated murine Supt4h and the human SUPT4H gene products are identical. The likely functional copy of SUPT4H, which has at least two introns, maps to human chromosome 17, with candidate intronless pseudogenes on chromosomes 2, 12, and 20. Buttressing the hypothesis that this is a gene required constitutively, both the human SUPT4H transcripts and the murine Supt4h transcript are expressed widely, although not at equal levels (e.g., such as most histones), in all fetal and adult tissues that we examined.

Adult↗
PubMed1996

Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.

We present a fluorescent-PCR-based technique to assay genomic sequence copy number and transcriptional abundance. This technique relies on the ability to follow fluorescent PCR progressively in real time during the exponential phase of the reaction so that quantitative PCR is accomplished. We demonstrated the ability of this technique to quantitate both known deletions and amplifications of loci that have been measured previously by other methods, and to measure transcriptional abundance. Using an efficient variant of the fluorescent-PCR technology, we can monitor transcription semiquantitatively. The ability to detect all amplifications and deletions at any single copy locus by PCR makes this the technique of choice to assay genomic sequence copy number anomalies in birth defects and cancers. The ability to detect variations in transcript abundance enables this technique to fashion a time and tissue analysis of transcription.

Chromosome Aberrations↗
PubMed1995

Expressed sequence tags from the long arm of human chromosome 21.

We isolated expressed sequence tags (ESTs) on the long arm of chromosome 21. The ESTs were mapped by PCR using a monochromosomal somatic-cell mapping panel. Of a total of 55 cDNAs, 30 mapped back uniquely to chromosome 21, 7 mapped back to other chromosomes including chromosome 21, 8 mapped back to chromosomes other than 21, and 10 could not be assigned using this methodology. The 30 chromosome 21-specific markers so isolated represent useful EST markers. A rapid PCR-based method was used to delineate the expression pattern of these 30 pairs in different tissues.

Animals↗
PubMed1995

Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis.

We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was > or = 650 base pairs and the average initial complexity of the given libraries was 10(6). We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development.

Base Sequence↗
PubMed1995

Transcription patterns of sequences on human chromosome 21.

The polymerase chain reaction (PCR) was used to screen embryonic, fetal and adult human cDNA libraries for transcription on chromosome 21q22.1-->q22.3. Seventy-three pairs of oligonucleotide primers on chromosome 21, used previously to screen a fetal brain cDNA library, were applied to analyze 41 different cDNA libraries. Only phage eluate (and therefore no DNA isolation) was required for this sensitive screening. Sixty primer pairs were positive with at least one cDNA library, indicating that the majority of primers were derived from transcribed sequences. Even with our most complex human fetal brain cDNA library, we detected only 57% (34/60) of transcribed sequences, illustrating the need to screen multiple human cDNA libraries to determine if transcription occurred. Since only 3/73 clones were present in only one cDNA library, the vast majority of transcribed sequences are present in more than one tissue.

Chromosomes, Human, Pair 21↗
PubMed1993

Expression of the IV (reversed and/or heterotaxic) phenotype in SWV mice.

Approximately 50% of iv/iv mice have situs inversus (mirror image reversal of viscera) and 40% have heterotaxia (anomalous arrangement of viscera). The occurrence of heterotaxia is independent of situs. Using the cross-intercross breeding system to put the iv gene on the SWV background, an occasional presumed iv/+ mouse was found that had an IV (situs inversus and/or heterotaxic) phenotype. Testcrosses of these reversed animals indicated an iv/+ genotype. Since iv is linked tightly to Igh-C on chromosome 12, we inferred the genotype with a polymorphism of Igh-C demonstrated using the polymerase chain reaction (PCR). This confirmed them to be iv/+. The expression of the IV phenotype in animals heterozygous for the iv gene may be due to an interaction of iv with an autosomal recessive gene found in SWV. We have not found the IV phenotype in heterozygous iv/+ mice following placement of the iv gene on six other inbred strains. Rarely, we also found that presumed SWV +/+ mice had the IV phenotype. Test matings of these phenodeviants, corroborated by PCR, have confirmed them to be +/+. Although the phenotypes of the affected SWV +/+ and iv/+ mice resembled those found in iv/iv mice, the occurrence of situs inversus and heterotaxia were not independent of each other, and most of the SWV mice with the IV phenotype had heterotaxia with situs solitus. This infrequent dominant expression of the iv gene has so far only been seen when iv is on the SWV background. These findings are consistent with the idea that this phenomenon is due to the interaction of the iv gene with another gene found so far only in the SWV strain.

Animals↗
PubMed1993

A recombination-based assay demonstrates that the fragile X sequence is transcribed widely during development.

To identify transcribed sequences rapidly and efficiently, we have developed a recombination-based assay to screen bacteriophage lambda libraries for sequences that share homology with a given probe. This strategy determines analytically whether a given probe is transcribed in a given tissue at a given time of development, and may also be used to isolate preparatively the transcribed sequence free of the screening probe. We illustrate this technology for the fragile X sequence, demonstrating that it is transcribed ubiquitously in an 11 week fetus, in a variety of 20 week human fetal tissues, including brain, spinal cord, eye, liver, kidney and skeletal muscle, and in adult jejunum.

Adult↗
PubMed1992

Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones.

We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.

Bacteriophage lambda↗
PubMed1992

A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].

Base Sequence↗
PubMed1992

Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21.

Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid beta protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.

Animals↗
PubMed1991

Plasmids for recombination-based screening.

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.

Bacteriophage lambda↗
PubMed1991

Developmental appearance of proteins identified by two-dimensional gel electrophoresis in mouse gonadal tissue.

Gonadal protein patterns of the mouse were studied during fetal development by two-dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post-coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.

Animals↗
PubMed1991

Analysis of proteins expressed at the time of murine organogenesis.

Two-dimensional electrophoretograms were prepared from wild-type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed in mouse embryos development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament-associated protein and tropomyosin-4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.

Animals↗
PubMed1991

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy).

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.

Animals↗