Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones.     

Chromosome 5 allele loss at the glucocorticoid receptor locus in human colorectal carcinomas

Matched normal/tumor DNA pairs from sporadic colon carcinoma patients were examined for chromosome 5 allele loss using a probe for a functional gene (glucocorticoid receptor = GRL) locus. This locus maps (5q11-q13) close to one of two alternative sites recently reported for a constitutional deletion in a familial adenomatous polyposis (FAP) patient. Tumor-specific allele loss of at least 27% at GRL supports the hypothesis that both hereditary and sporadic forms of colon cancer result from mutations of the same gene. The proximity of the GRL locus to the region of 5q affected in FAP and the observed tumor-specific allele loss at this locus suggest that further research is needed regarding whether genetic alterations in the glucocorticoid receptor may be associated with colon carcinogenesis.     

Bacteriophage-resistant mutants of Bacillus thuringiensis with decreased virulence in pupae of Hyalophora cecropia

Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia). These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella. They were also more sensitive to cecropia immune hemolymph in vitro. In addition, the export of at least three proteins was reduced. Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives. One class II mutant was a complete revertant in all properties examined. The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins. Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent. Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin.     

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Mutant of Escherichia coli with Anomalous Cell Division and Ability to Decrease Episomally and Chromosomally Mediated Resistance to Ampicillin and Several Other Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain, we isolated two smooth mutants which were both sensitive to ampicillin and carried defects in the cell envelope. One of the strains (with the envA gene) is hindered in its completion of septa and forms chains of cells. The envA gene has been mapped to a position between leu and proB, at 2 to 4 min. The envA gene decreased the resistance mediated by both episomal and chromosomal genes for resistance to several antibiotics. During growth the envA mutant was characterized by abnormal ratios between viable count or cell count and optical density. The ratio between viable count and optical density was affected during shift-up and shift-down experiments. When compared to the parent strain, the envA mutant was found to be more resistant to ultraviolet irradiation on plates. Prestarvation for tryptophan had a protective effect against irradiation both on the parent strain and the envA mutant.     

Lack of transmission of deleted mtDNA from a woman with Kearns-Sayre syndrome to her child.

We have investigated the daughter of a woman with Kearns-Sayre syndrome. The woman had a high percentage of deleted mtDNA in muscle, but no deleted mtDNA was detected in fibroblasts, bone marrow, and peripheral blood cells by Southern blot analysis. With PCR, analytical sensitivity was significantly increased, and deleted mtDNA was detected in all examined tissues from this patient. The patient had healthy parents and nine healthy siblings. No deleted mtDNA was detected in blood from the mother of the patient. The patient had an uneventful pregnancy and delivered at term. Deleted mtDNA could not be detected in placenta by Southern blot analysis. With PCR, deleted mtDNA was detected in the majority of placental specimens. This finding may, however, be due to contamination with maternal DNA. The patient's daughter was healthy at age 5 mo, and morphologic examination of muscle was normal. No transmission of deleted mtDNA to the daughter could be detected by Southern blot and PCR analysis of peripheral blood cells, bone marrow, fibroblasts, and muscle. The presence of deleted mtDNA was excluded at a fractional level of less than 1:100,000 in all examined tissues from the daughter.     

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.