An intracisternal A-particle DNA sequence is closely linked to the thrombomodulin gene in some strains of laboratory mice.

Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a difference in two strains of laboratory mice, BALB/cBD (maintained at the Oak Ridge National Laboratory) and the strain used for the sequence listed in the GenBank data base. An intracisternal A-particle (IAP) provirus was present in the BALB/cBD sequence but absent from the GenBank sequence. Thus, there are two different 5' regions for the TM gene in mice.

Contributions of cooling and warming rate and developmental stage to the survival of Drosophila embryos cooled to -205 degrees C.

Because of their high susceptibility to chilling injury, permeabilized Drosophila embryos can not be cryobiologically preserved by slow freezing at rates low enough to prevent the formation of intraembryonic ice. Calculations indicated that to outrun the chilling injury they must be cooled and warmed rapidly at an estimated 20,000 degrees C/min or faster. Ordinarily, such cooling rates would inevitably produce lethal intracellular ice.

Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA.

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme.

Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF.

Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule).

Critical factors affecting the permeabilization of Drosophila embryos by alkanes.

Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum.

Thrombomodulin is preferentially expressed in Balb/c lung microvessels.

Previously, two rat monoclonal antibodies where developed which bind distinct epitopes on a murine glycoprotein, P112, which is expressed primarily in lung capillary endothelium. In this paper we show that P112 is identical to the endothelial anticoagulant protein, thrombomodulin (TM). Several lines of evidence support this conclusion.

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally.

Murine fumarylacetoacetate hydrolase (Fah) gene is disrupted by a neonatally lethal albino deletion that defines the hepatocyte-specific developmental regulation 1 (hsdr-1) locus.

Homozygous deletion of the hepatocyte-specific developmental regulation 1 (hsdr-1) locus in mouse chromosome 7 results in perinatal death and a pleiotropic syndrome characterized by ultrastructural abnormalities of the liver and kidney, failure of induction of a number of specific transcription units in the liver and kidney during late gestation, and marked overexpression of an enzyme that defends against oxidative stress. Previously, the breakpoints of two albino (c) deletions (c14CoS and c1FAFyh) that genetically define hsdr-1 were localized, on a long-range map, in the vicinity of the distal breakpoint of a viable albino deletion (c24R75M) that breaks proximally within the c locus. Here we report the use of a probe derived from a deletion breakpoint fusion fragment cloned from c24R75M/c24R75M DNA to clone a breakpoint fusion fragment caused by the c14CoS deletion.

Characteristics and kinetics of subzero chilling injury in Drosophila embryos.

Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and -25 degrees C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, -9, and -15 degrees C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h.

Isolation and characterization of a 1-beta-D-arabinofuranosylcytosine-resistant Chinese hamster ovary cell mutant that is also X-ray sensitive and is noncomplementary with ataxia telangiectasia cells.

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair.

Basal cells are the progenitors of primary tracheal epithelial cell cultures.

The goal of this study was to identify the cells from the rat tracheal epithelium which attach and proliferate in primary culture. When cells isolated from tracheas by enzymatic digestion were held in suspension at 37 degrees C for several hours most of the differentiated cells died. The kinetics of this selective cell death were not dependent on the constituents of the holding medium.

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase.

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli.

Mutational analysis of leucine 47 in human epidermal growth factor.

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity.

Structure of the macronuclear polyubiquitin gene in Euplotes.

The hypotrichous ciliate, Euplotes eurystomus, contains both a transcriptionally inactive micronucleus (MIC) and a transcriptionally active macronucleus (MAC) in the same cell. MAC DNA is small (0.5-20 kb), linear and highly amplified.

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis.

Female-specific mutagenic response of mice to hycanthone.

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division.

Physicochemical studies of human O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.

Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding.

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species.

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification.

Uptake and metabolism of putrescine in confluent LLC-PK1 cells.

Putrescine is taken up by confluent pig kidney (LLC-PK1) cells at roughly equal rates over both Na(+)-dependent and Na(+)-independent pathways. The former is sensitive to 1 mM amiloride, but the latter is not. Uptake rates are similar at both the apical and basolateral surfaces.

The aggregation states of spinach phosphoribulokinase.

Phosphoribulokinase (PRK; EC 2.1.7.