Assignment of human pepsinogen C (PGC) gene to chromosome 6.

cDNA of rat pepsinogen C (PGC) hybridizes to, among others, a 3.2-kb band in Southern blot analysis of BamHI-cleaved human genomic DNA. This property was employed to localize the human PGC gene. Use of flow-sorted human chromosomes and 12 human x mouse somatic cell hybrid lines demonstrated that the gene is located on chromosome 6.

Primary structure of human pancreatic secretory trypsin inhibitor (PSTI) gene.

The human pancreatic secretory trypsin inhibitor (PSTI) gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is approximately 7.5 kb long and is separated into four exons by three introns. The gene has multiple transcription start points and examination with a single-laser cell-sorter showed that it is located on chromosome 5.

Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.

Human gene coding for granulocyte-colony stimulating factor is assigned to the q21-q22 region of chromosome 17.

Granulocyte-colony stimulating factor (G-CSF) is a member of colony stimulating factors which regulate the proliferation and differentiation of hematopoietic progenitor cells. A full-length cDNA clone coding human G-CSF was used as a hybridization probe to detect homologous sequence in human-mouse somatic cell hybrids, flow-sorted human chromosomes, and in situ human metaphase chromosomes. The results indicate that the gene encoding human G-CSF is on the q21-q22 region of chromosome 17, which is involved in translocation of t(15;17) (q23;21) in human acute promyelocytic leukemia.

Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation.

The human RCC1 gene was cloned after DNA-mediated gene transfer into the tsBN2 cell line, which shows premature chromosome condensation at nonpermissive temperatures (39.5-40 degrees C). This gene codes for a 2.5-kb poly(A)+ RNA that is well conserved in hamsters and humans. We isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone. The base sequences of these two active cDNA clones differ at the 5' proximal end, yet both have a common open reading frame, encoding a protein of 421 amino acids with a calculated molecular weight of 44,847 and with seven homologous repeated domains of about 60 amino acids. This human RCC1 gene was located to human chromosome 1 using sorted chromosomal fractions.

Replication of mini-F plasmid in vitro promoted by purified E protein.

An in vitro system for replication of mini-F plasmid DNA was constructed. This system consists of an ammonium sulfate fraction II (Fuller et al. 1981) from Escherichia coli extract, exogenously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form. Experiments with this system showed that the 217 bp DNA region which contains the A + T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication.

Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.

Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication. We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity. Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene. These binding sites have a common 8-base-pair sequence. These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control. They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.

Chromosomal assignment of human genes for gastrin, thyrotropin (TSH)-beta subunit and C-erbB-2 by chromosome sorting combined with velocity sedimentation and Southern hybridization.

Human genes for gastrin, thyrotropin (THS)-beta subunit and c-erbB-2 were assigned to specific chromosomes using a single-laser cell sorter. For this purpose, condensed human chromosomes prepared from a karyotypically normal lymphoblastoid cell line were preliminarily fractionated by velocity sedimentation, and then sorted using a fluorescence-activated cell sorter. DNA was then extracted from the chromosomes, cleaved by restriction enzymes, and subjected to Southern hybridization using gene-specific radioactive probes. When the assignment of specific chromosomes was not possible due to chromosomal size overlapping, sorted chromosomes from cell lines carrying chromosomal translocation or from hybrid cells carrying known human chromosomes were used in addition. The results indicate that human genes for gastrin, TSH-beta, and c-erbB-2 are located on chromosomes 17, 1 and 17, respectively.

Structure of human cholecystokinin gene and its chromosomal location.

We have determined the entire structure of human cholecystokinin (CCK) gene, which is 7 kb in size and separated into three exons. S1 endonuclease analysis has shown two putative transcription initiation sites that are preceded by 'TATA' equivalent sequences located 39 bp and 35 bp upstream from these sites. The promoter region contains five 'G-C box'-like sequences, which are believed to be sp 1-binding sites. By chromosome sorting in combination with velocity sedimentation and Southern hybridization, the human cck gene was mapped on the short arm of human chromosome 3.

Mini-F protein that binds to a unique region for partition of mini-F plasmid DNA.

A mini-F-coded protein, named F2 protein, binds specifically to mini-F DNA. This protein has a molecular weight of 37,000 and is coded by the A2 segment of the mini-F genome (47.3 to 49.4 kilobases on the F coordinate map). The binding site is located also in the A2 segment of mini-F. This binding site is lost by spontaneous deletion when the A2 segment alone, but not A2 together with its neighboring segment, is cloned in a multicopy plasmid pBR322. These data are discussed in connection with incompatibility and plasmid stability.

Identification of the minimal essential region for the replication origin of miniF plasmid.

The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with hat of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).

A new packing for separation of DNA restriction fragments by high performance liquid chromatography.

TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.

Operational variables in high-performance gel filtration of DNA fragments and RNAs.

Double-stranded DNA fragments and ribosomal and transfer RNAs were measured by high-performance gel filtration on TSK-GEL G2000SW, G3000SW, G4000SW and G5000PW columns to investigate the separation range and resolution of these columns and the effects of eluent ionic strength and flow-rate on retention and resolution. These columns could separate double-stranded DNA fragments up to ca 1 X 10(6) and rRNAs up to ca 5 X 10(6) daltons in molecular weight. However, it was found that the selection of the column is very important to achieve optimum separation, depending on the molecular weight of the sample. Elution is delayed as the eluent ionic strength is increased. An eluent ionic strength of 0.3-0.5 seemed appropriate in most cases. Resolution is greatly increased as the flow-rate is decreased.