Characterization of the central region containing the X-inactivation center and terminal region of the mouse X Chromosome using irradiation and fusion gene transfer hybrids

The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.     

Teratogenic effects of parthenogenetic cells from LTXBO mice,a strain which develops ovarian teratomas at high frequency

Summary LTXBO mice develop ovarian teratomas at high frequency. The phenotype of tumour tissues is unusual in that most contain trophoblast elements. Since the tumours are derived from parthenogenetically activated oocytes, they would not be expected to produce trophoblast. The developmental potential of parthenogenetic cells from these mice was tested in aggregation chimeras. No contribution to trophoblast tissues was observed. However, a high incidence of morphological abnormalities was seen, suggesting that the parthenogenetic cells exerted a teratogenic effect.     

The human genetic map

The introduction of new technology and increased effort from around the world is driving the completion of the human gene map. In parallel with the creation of the map, we are beginning to see the bio-medical benefits that are a direct consequence of learning more about our own genome.     

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.     

Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity.

Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C. A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C. These findings suggested a possible linkage between methionine metabolism and heat shock. We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation. Exponential-phase cultures of the two strains were pulse-labeled with [3H]leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis. The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed. Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains. Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain. The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R. A. VanBogelen, M. A. Acton, and F. C. Neidhardt, Genes Dev. 1:525-531, 1987). We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.     

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R.T., & Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766. Fujii, K., & Huennekens, F.M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of the basement membrane of human lymph node high endothelial venules: An ultrastructural, histochemical and immunocytochemical study

Summary The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase,-glucuronidase,-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.     

Thermodynamic analysis of the lactose repressor-operator DNA interaction

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.     

Transient modulation and internalization of T4 antigen induced by phorbol esters

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.