PubMed Health⌕ Search

Biomedical subjects

K Osoegawa

Publications and source records attributed to K Osoegawa.

26 records · Page 2Linked to original sources
PubMed1998

Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Animals↗
PubMed1996

A 19-kb CpG island associated with single-minded gene 2 in Down syndrome chromosomal region.

To help in isolating the genes involved in Down syndrome, we sought CpG islands in 4 Mb cosmid/PAC contigs spanning most of the 21q.22.2 band using seven rare cutting enzymes. A striking feature was observed upstream of hSIM2 where at least 41 rare-cutting sites were clustered within a 20-kb region. To investigate the structure of the cluster, a cosmid containing hSIM2 was submitted to shotgun sequencing. Sequence analysis revealed that the cluster was a long CpG island extending 19, 128 nucleotides which includes in the first and second exons of hSIM2. Taken together with our observation in which the CpG islands were concentrated within 1.2 Mb around hSIM2, we propose that this region functions as an R-band, and the cluster provides a unique element for marking of DNA for the spatial and temporal expression of the hSIM2 locus.

Amino Acid Sequence↗
PubMed1996

An integrated map with cosmid/PAC contigs of a 4-Mb Down syndrome critical region.

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.

Bacteriophage P1↗
PubMed1996

Isolation of a cosmid clone corresponding to an inv(21) breakpoint of a patient with transient abnormal myelopoiesis.

Transient abnormal myelopoiesis (TAM) is a leukemoid reaction occurring occasionally on Down syndrome (DS) newborn infants. It has been hypothesized that "disomic homozygosity" in 21-trisomic cells plays an important role in the genesis of TAM, and the putative TAM gene was suggested to be mapped at a 21q11 region. We encountered a DS-associated TAM infant with a 47,XY,inv(21)(q11.1q22.13),+inv(21)(q11.1q22.13) karyotype. On the basis of another presumption that in this patient the putative TAM gene is disrupted by the break, we tried to isolate a breakpoint DNA. FISH analysis with cosmid clones corresponding to various sequence-tagged-site (STS) markers mapped at around 21q11.1-q11.2, we confirmed that the proximal breakpoint of the inv(21) was located between two STSs, G51E07 and D21S215, the latter locus being consistent with the previous tentative mapping. After construction of a cosmid contig encompassing between the two markers, we have isolated a cosmid clone corresponding to the proximal breakpoint of the inversion. This breakpoint was located near a previously identified duplicated region that is homologous to the sequence at 21q22.1. The isolated cosmid clone is useful for analysis of other TAM patients and for a search for a transcript at or flanking the breakpoint.

Adult↗
PubMed1995

Cosmid assembly and anchoring to human chromosome 21.

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Animals↗
PubMed1994

Streptomyces ATP nucleotide 3'-pyrophosphokinase-gene cloning and sequence analysis.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

Amino Acid Sequence↗
PubMed1992

Streptomyces ATP nucleotide 3'-pyrophosphokinase and its gene.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular, ribosome-independent, and stringent factor-mimic ppGpp synthetase with an unusually broad acceptor spectrum. The gene-containing DNA fragments cloned from chromosomal DNA of a producer S. morookaensis into pIJ699 and pUC plasmids were found to express the active enzyme in the transformed S. lividans TK24 and enteric E. coli JM109 and nitrogen-fixing Klebsiella pneumoniae M5a1 and 5022, respectively. Base sequence of the structural gene and the deduced amino acid sequence exhibited little homology to those of E. coli stringent factor and related proteins. Growth retardation was seen in some transformants.

Base Sequence↗