Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.     

Measurement of oxidation-reduction midpoint potentials by room temperature electron paramagnetic resonance potentiometry

A room temperature electron paramagnetic resonance potentiometric cell has been developed for the measurement of oxidation-reduction midpoint potentials of enzymes containing paramagnetic centers. Based upon an aqueous flat cell designed for use with the Varian TM high sensitivity cavity, the apparatus combines a high degree of anaerobiosis with low volume requirements. The cell is simple in design, easily constructed, and can be adapted for use with most spectrometer cavities. Tests of the cell using xanthine oxidase, in 50 mM Bicine buffer, pH 7.7, yielded midpoint potentials of -345 and -371 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples compared with values of -373 and -377 mV obtained by electron paramagnetic resonance analysis of frozen potentiometric samples. These values indicate that shifts, of the order of 20-40 mV, may occur upon freezing poised samples. For the Mo center of xanthine oxidase, these shifts in potential are more pronounced for the Mo(VI)/Mo(V) couple and result in a destabilization of the Mo(V) intermediate during freezing.     

New roles for DIF? Effects on early development in Dictyostelium

The DIFs are unusual, chlorinated molecules which induce stalk cell differentiation during the later, multicellular phase of Dictyostelium development. Here we provide evidence that one or more DIFs have a role during early development, when small amounts are known to be made. Initial indications came from an optical technique which detects changes in shape or cohesion of cells in suspension (Gerisch and Hess, PNAS 71, 2118, 1974). After a period of optical inactivity at the start of development, cell suspensions normally produce spontaneous spike-shaped light-scattering oscillations synchronised by oscillations in extracellular cAMP levels, followed by sinusoidal oscillations where the synchroniser is not known. DIFs 1 and 2 produce optical responses from cells at all these early stages of development. The phase of both spiked and sinusoidal oscillations can be shifted, indicating an effect on the oscillator in each case. We find further: (1) cAMP oscillations and cAMP relay during spiked oscillations are transiently inhibited by DIF-1. (2) DIF-1 causes a transient decrease in cellular cGMP levels in cells taken before oscillations commence and likewise inhibits the cGMP response to a cAMP stimulus in cells taken later in development. Cytoskeletal organization and hence cell shape might be affected by DIF-1 by this indirect route. (3) The effects of DIF-1 are transient, even though it is essentially stable in the cell suspension. Cells somehow adapt to DIF-1. (4) The effects are chemically specific: DIF-1 and DIF-2 are active at 10(-7) to 10(-8) M, with DIF-2 being the more active, whereas related compounds have little or no activity at 10(-6) M. These results indicate that cells are responsive to DIFs 1 and 2 from the start of development and suggest a wider role for the DIFs. This role might involve effects on cAMP signalling and on intracellular second messengers.     

Degradation of myofibrillar proteins by trypsin-like serine proteinases.

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.     

The mendelian inheritance of a human X chromosome-specific DNA sequence polymorphism and its use in linkage studies of genetic disease

Summary A recombinant DNA sequence, RB6, was isolated from a human X chromosome library and shown to be X-specific by hybridisation to DNA from a human-mouse somatic cell hybrid containing X as the only human chromosome. The cloned sequence was located on the long arm distal to Xq13 using a human-mouse somatic cell hybrid containing a partial human X chromosome. DNA samples isolated from control human females were digested with the restriction enzyme MspI, and analysed by blotting and hybridisation to the radioactive cloned DNA. Eight of 14 individuals from a random population showed a single hybridising band 7.5 kilobase pairs (kb) in length, but six showed an additional band 10.1 kb in length. DNA from 12 members of a family with X-linked thyroxine-binding globulin deficiency was analysed for the segregation of this polymorphism. The results show that the polymorphism is inherited in a Mendelian fashion, and that the disease locus is not closely linked to the polymorphic site. Such polymorphisms will be useful as markers for chromosome mapping and for the antenatal diagnosis of genetic diseases.     

The ontogeny of premolar dental wear in Cercocebus albigena (cercopithecidae)

The orientation of striated wear facets on primate teeth serves as a useful guide for reconstructing jaw movements during mastication. Most wear facets on the molars are formed during one of the two well-documented movements, Phase I or Phase II, of the power stroke. Another jaw movement direction, “orthal retraction” (OR) has been proposed to account for a third set of facets occasionally present on the pointed tips of premolars and molars. Evidence advanced here indicates that OR facets on pointed anterior premolars (P₃) of cercopithecoids are actually Phase I facets that have become reoriented as a result of a rotation of this tooth during its eruption. “Orthal retraction” probably does not exist as a discrete masticatory phase.     

Increased Noradrenergic Metabolism in the Cerebellum of the Mouse Mutant Dystonia Musculorum

Abstract: The neurological mouse mutant dystonia musculorum exhibits bizarre appendicular and truncal dystonia without known cerebellar histopathology. We evaluated striatal dopamine and cerebellar norepinephrine metabolism in this mutant and compared the results with those obtained in wild-type BALB/c and B6C3 controls. Tyrosine hydroxylase activity and dopamine metabolite levels (homovanillic acid and 3,4-dihydroxyphenylacetic acid) in the striatum of the mutant were similar to controls. Tyrosine hydroxylase activity and the steady-state level of 3-methoxy-4-hydroxyphenethyleneglycol, a metabolite of norepinephrine, in the cerebellum were 38% and 42-66%, respectively, greater in the mutant. However, the level of norepinephrine was similar (∼350 ng/g). Further, a Purkinje cell-specific marker, cGMP-dependent protein kinase, was unchanged in the mutant and no Purkinje cell pathology was observed with light microscopy. The lack of Purkinje cell derangement and similar levels of cerebellar norepinephrine and cGMP-dependent protein kinase activity suggest that increased norepinephrine metabolism in the cerebellum of this mutant is not a morphological response to gross target cell loss during morphogenesis. The observed changes may be a reaction to abnormal impulse traffic or altered input/output pathways to the mutant cerebellum during its development.