Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.     

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.     

Catalase-like crystals in parathyroid gland cells of Rana temporaria L.

Summary Crystalline inclusions in parathyroid gland cell nuclei of Rana temporaria were studied by electron microscopy using a specimen tilting stage. Images were analysed by optical diffraction. Results were compared with X-ray and electron microscopic data of trigonal bovine liver catalase to which a striking resemblance of the inclusions was found.We are grateful to Professor R. Mosebach (Giessen) for discussions, to the Deutsche Forschungsgemeinschaft for a grant (La 229/4) and instruments and to Messrs. Spindler & Hoyer, Göttingen and Messrs. Rank Precision Instruments, Nürnberg for putting apparatus at our disposal and performing diffraction photographs.     

Location of phosphorylase kinase (Phk) in the mouse X chromosome

The phosphorylase kinase deficiency (Phk) locus has been located in the mouse X chromosome, the order of genes being centromere-Bn-Phk-Ta-jp. Since the Phk locus of the mouse may be identical to the locus responsible for the X-linked phosphorylase kinase deficiency trait of man, and there may be a high degree of gene-order homology in the X chromosome of all mammals, the location of Phk in the mouse reported here may aid in locating the phosphorylase kinase gene on the X chromosome of man.This research was supported by grants AM 13359 (to F.H.) and AM 14461 (to D.L.C.) from the National Institute of Arthritis and Metabolic Diseases, and by an allocation (to E.M.E.) from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. F.H. is a recipient of a Research Career Development Award (AM 46 421) of the National Institute of Arthritis and Metabolic Diseases.     

Linkage analyses using biochemical variants in mice. II. Levulinate dehydratase and autosomal glucose 6-phosphate dehydrogenase

The level of hepatic -aminolevulinate dehydratase varies among inbred strains of mice and is regulated by codominant alleles at the Lv locus. Twenty-two inbred strains have been classified with respect to this locus. Lv is 5±2 recombination units from brown, b, in linkage group VIII. The locus for autosomal glucose 6-phosphate dehydrogenase (Gpd-1) has also been assigned to linkage group VIII and is 32±5 units from brown. The order of the loci is Lv-b-Gpd-1. Incidental note is made of linkage between the malic dehydrogenase (Mdh-1) and dilute (d) loci, linkage group II, with 10±3 % recombination between the two.Supported by the Roche Institute of Molecular Biology, Nutley, New Jersey, and by Public Health Service Research Grant CA-05873 from the National Cancer Institute.     

BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.