Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

A novel actin label: a fluorescent probe at glutamine-41 and its consequences

By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Changes in Microtubules in Onion Leaf Sheath Cells during Bulb Development

Cortical microtubules are oriented transversely to the cellaxis in leaf sheath cells of onion plants (Allium cepa L. cv.Osaka-Okute) that have not started bulb formation. As the bulbdevelops and the leaf sheath cells swell, the microtubules becomedisoriented and scattered and finally disappear. The microtubuleinhibitors colchicine and cremart [O-ethyl O-(3-methyl-6-nitrophenyl)N-sec-butylphosphorothioamidate]cause swelling of leaf sheath cells and make the basal partof the plant bulbous. The cortical microtubules may have animportant role in regulating bulb development in onion plants. (Received August 21, 1982; Accepted December 6, 1982)     

Clinical Use of a New Mitral Disc Valve

A disc valve of new design was used successfully for the replacement of the mitral valve in patients with rheumatic mitral valve disease. This valve would appear to have the following advantages over the mitral ball valve prosthesis:• Lower left atrial pressure after replacement.• Elimination of the hazard of left ventricular outflow tract obstruction with mitral valve replacement.• Decreased incidence of thromboembolization.• Abolition of possibility of ventricular septal irritation.Despite the better outlook for this valve compared with the ball valve for mitral valve substitution, the mitral valve should always be repaired whenever feasible. Repair is possible in the majority of patients.