Transcription of cloned tRNA gene fragments and subfragments injected into the oocyte nucleus of Xenopus laevis. Cloned 3.18 kilobase fragments of Xenopus laevis DNA containing genes for tRNAMet1 and for at least one other 4S RNA species are transcribed rapidly after their injection into the nucleus of X. laevis oocytes. The newly synthesized RNA can be resolved by gel electrophoresis into a few predominant 4S RNA species and a series of slower migrating components. One of the 4S RNA species appears to be identical, by fingerprint analysis, to the tRNAMet1 isolated by hybridization of somatic cell RNA to this cloned tRNA gene fragment (tDNA). Thus, the tRNAMet1 produced after injection can be both fully processed and modified. Its rate of synthesis is estimated to be about 6 x 10(9) molecules/hr in each oocyte injected with 2 ng of tDNA. When the tDNA fragment is cleaved into two halves with restriction endonuclease Sst I, each injected half gives rise to a subset of the RNAs produced after injection of the intact fragment. This experiment thus suggests the presence of at least two transcriptional units on this cloned tDNA. This simple way of biologically testing defined restriction fragments may be of value for analyzing the functional organization of other cloned eukaryotic DNA units.


Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts. High D/L aspartic acid ratios are observed in heavy molecular weight aggregates and in water-insoluble protein extracted from whole lenses and nuclear and cortical regions. Purified alpha-, beta-, and gamma-crystallins have low D/L ratios. Fractionation of urea-solubilized material from the water-insoluble protein yields four molecular weight classes of proteins. Fractions representing crosslinked material or apparently degraded products have high D/L ratios. Racemization within lens proteins may contribute to formation of the water-insoluble fraction seen in aging lenses and cataracts.


Ligandin retains and albumin loses bilirubin binding capacity in liver cytosol. Circular dichroism methods were used to detect bilirubin-ligandin interactions in rat liver cytosol and fractions obtained at various stages during purification of ligandin. Ligandin retained its capacity to bind bilirubin in the presence of components of liver supernatant, but albumin, which binds bilirubin in serum, lost the capacity to bind bilirubin in liver supernatant. This was attributed to a greater binding specificity exhibited by ligandin. In their respective physiological milieus, albumin and ligandin are structurally adapted to bind ligands: albumin in serum, and ligandin in the cytosol of the liver cell. These studies are consistent with the hypothesis that the concentration of ligandin within the liver could regulate the net flux of certain organic anions from plasma into the liver.


Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis. Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA in vitro. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C(0)t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.


Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders. Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [(32)P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained beta-like gene sequences assayed with beta globin cDNA probe. One beta-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three beta gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing beta-like genes were absent from deltabeta thalassemic DNA and one new fragment containing beta-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of beta-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with deltabeta thalassemia.


Cardiolipin: a stereospecifically spin-labeled analogue and its specific enzymic hydrolysis. The spin-labeled cardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-(16-doxylstearoyl)glycero(3)phosphol]-sn-glycerol has been prepared. The stereoselective synthesis makes use of the monolysocardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-lyso-sn-glycero(3)phospho]-sn-glycerol, available from the stereospecific hydrolysis of cardiolipin by phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of Trimeresurus flavoviridis. The results of treatment of the spin-labeled cardiolipin with the cardiolipin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) (Hemophilus parainfluenzae) of known specificity and with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) of Bacillus cereus are consistent with the assigned structure. The spin-labeled cardiolipin is further characterized and the unique features of this diastereomer are discussed in the context of the unusual stereochemistry of the natural phospholipid.


Defective regulation of cholesterol biosynthesis and plasma membrane fluidity in a Chinese hamster ovary cell mutant. A Chinese hamster ovary cell mutant resistant to killing by 25-hydroxycholesterol is shown to be defective in the regulation of cholesterol synthesis by exogenous cholesterol. When grown with various cholesterol supplements in delipidized serum, the mutant cell exhibits a variation in cholesterol content and plasma membrane fluidity that is not observed in the parental cell type.


Addition of mononucleotides to oligoribonucleotide acceptors with T4 RNA ligase. RNA ligase induced by bacteriophage T4 catalyzed the addition of nucleoside 5',3'-diphosphates to oligoribonucleotide acceptors in the presence of ATP. The reactions proceeded in equimolar concentrations of donors and acceptors. Several oligonucleotides of defined sequence were synthesized by this method in yields of 28-68%. The enzyme required the phosphate ester at the 3' position of the donor molecule, nucleoside 5',2'-diphosphates being unable to serve as donors. Thymidine 5',3'-diphosphate was active as a donor for the enzyme. The ligation product, (Ap)2ApCp, was also obtained from the reaction of (Ap)2A and 5'-adenylylated cytidine 5',3'-diphosphate (A5'pp5'Cp) with RNA ligase in the absence of ATP. These results show that the minimal substrate for RNA ligase is a nucleoside 5',3'-diphosphate.


DNA polymerase activities in differentiating mouse neuroblastoma N-18 cells. The activities of two DNA polymerases (DNA nucleotidyltransferases) were characterized in mouse neuroblastoma clone N-18 on the basis of their apparent molecular weights (determined by sucrose density gradient centrifugation: polymerase-alpha, 7.5-8 S; polymerase-beta, 3-4 S) and relative inhibition by sulfhydryl-blocking agents. N-Ethylmaleimide (10 mM) and iodoacetamide (1.5 mM) inhibited DNA polymerase-alpha activity completely, whereas only 35-40% inhibition was observed for DNA polymerase-beta under similar conditions. DNA polymerase-alpha activity was reduced 50-70% in N-18 cells that had been induced to differentiate by 4 micro M bromodeoxyuridine, and the low molecular weight DNA polymerase-beta activity remain unchanged. With activated calf thymus DNA as template, only DNA polymerase-alpha activity was stimulated in the presence of added ribonucleotides and purified Escherichia coli RNA polymerase.


Intragenic DNA spacers interrupt the ovalbumin gene. We have performed restriction endonuclease mapping to examine the linear organization of the ovalbumin gene in chromosomal DNA. Treatment of genomic DNA with restriction endonucleases that do not cleave the ovalbumin mRNA sequence results in the generation of multiple DNA fragments capable of annealing with ovalbumin-specific probes in molecular hybridization reactions. These data strongly suggest that the linear order of DNA sequences coding for ovalbumin is interrupted by at least two intragenic DNA spacers absent from the corresponding RNA. At least one of these spacer sequences interrupts the coding sequence; therefore the chromosomal ovalbumin gene is not colinear with its translational product. We can discern no difference in the sequence organization about this gene in producer and nonproducer somatic cells, suggesting that the presence of intragenic spacers does not reflect that transcriptional activity of this gene. Furthermore, profiles obtained for gametes are identical to those observed for somatic cells, indicating that significant translocation during development is not responsible for the generation of this split sequence. Divergence of the intragenic spacer, however, is observed between individual chickens; thus multiple alleles may exist for this gene that are identifiable by differences in the organization of spacer with no apparent phenotypic evidence for their presence.


The intervening sequence of a mouse beta-globin gene is transcribed within the 15S beta-globin mRNA precursor. Mouse beta-globin in encoded in a discontinuous structural gene interrupted by a 550-base pair intervening sequence of DNA. Correspondingly, the mature beta-globin mRNA appears to be synthesized via a 15S precursor, the length of which roughly equals the total length of the coding and intervening sequences of the beta-globin gene. Using the electron microscope to visualize hybrid structures formed between this gene and the purified 15S beta-globin mRNA precursor, we show that the intervening sequence is present within the larger precursor molecule. This finding suggests that the precursor mRNA is processed through the removal and rejoining of internal RNA sequences.


Ligands containing heavy atoms: perturbation of phosphorescence of a tryptophan residue in the binding site of wheat germ agglutinin. Information on the structure of binding sites of wheat germ agglutinin was obtained on the basis of fluorescence and phosphorescence changes of tryptophan residues induced by the binding of several thiomercuribenzoate derivatives of glycosides. The thiomercuribenzoate derivatives bind selectively to wheat germ agglutinin in the same way as the corresponding sugars. Using the thiomercuribenzoate of di-N-acetyl-beta-chitobiose, it was found that: (i) the fluorescence of tryptophan residues was drastically quenched at both 298 and 77 K; (ii) the phosphorescence intensity was strongly enhanced at 77 K; (iii) the phosphorescence lifetime was markedly decreased. A similar effect was observed with the thiomercuribenzoate of N-acetyl-beta-D-glucosamine. These changes were completely reversed upon addition of 1-O-methyl-di-N-acetyl-beta-chitobioside. The thiomercuribenzoate of beta-D-glucose had no effect at all, and the thiomercuribenzoate of tri-N-acetyl-beta-chitotriose had a limited effect. These results are interpreted as a specific heavy atom effect due to a close contact between one tryptophan residue of the protein and the heavy atom of the bound ligand. They are consistent with the view that: (i) binding sites of wheat germ agglutinin may be divided in three subsites, A, B, and C; (ii) a tryptophan residue is in the binding site at subsite C; and (iii) this residue and the ligand are in close contact. This new method, using the enhancement of spin-orbit coupling due to the selective perturbation induced in a tryptophan residue by a ligand containing a heavy atom, has proved to be suitable for locating the tryptophan residue in the binding site of wheat germ agglutinin and can probably be extended to other sugar-binding proteins.


Evolution of the nervous system: role of ontogenetic mechanisms in the evolution of matching populations. Nervous systems are composed of populations of cells that are synaptically connected in a highly predictable manner, and we have called two interconnected populations a pair of matching populations. Heritable genetic changes that affect a pair of matching populations can be evolutionary only when this matching quality is not disrupted. We distinguish two types of heritable change. Concordant heritable changes autonomously preserve the match and are thus automatically candidates for what we call type I evolutionary change. Nonconcordant heritable changes, on the other hand, are those that do not autonomously preserve the match. Those nonconcordant heritable changes that can use other normally present ontogenetic mechanisms to preserve the match are candidates for what we call type II evolutionary change. One example of such an ontogenetic mechanism consists of the production of excess neuroblasts and the subsequent weeding out (via cell death) of those that do not successfully match. Because normal ontogeny is an integral part of type II evolutionary change, ontogenetic manipulations can give evolutionary insights. Embryonic graft experiments, in particular, can elucidate the nature of ontogenetic mechanisms that participate in type II changes. Thus, some developmental experiments can be considered to be evolutionary experiments.


Colony size distributions as a measure of in vivo and in vitro aging. Individual human diploid cells plated at low cell density and incubated for 2 weeks develop into colonies ranging in size from one cell to several thousand cells. The resultant colony size distribution is an accurate indicator of the number of subsequent in vitro population doublings that can be attained by the parent culture. This relationship holds for both human fetal lung and adult skin fibroblast cultures. In addition, the colony size distributions obtained from fetal, young adult, and old adult human cell cultures at the same low level of in vitro passage are indicative of the in vivo age of the level of in vitro passage are indicative of the in vivo age of the cell culture donor. Cell cultures of fetal origin give rise to the highest percentage of colonies with significant proliferative abilities, whereas cultures from old adults give rise to the lowest percentage of large colonies. Therefore, colony size distributions appear to be good indicators of both in vitro and in vivo human cellular aging.


Model for capping of membrane receptors based on boundary surface effects. Crosslinking of membrane surface receptors may lead to their segregation into patches and then into a single large aggregate at one pole of the cell. This process is called capping. Here, a novel explanation of such a process is presented in which the membrane is viewed as a supersaturated solution of receptors in the lipid bilayer and the adjacent two aqueous layers. Without a crosslinking agent, a patch of receptors that is less than a certain size cannot stay in equilibrium with the solution and thus should dissolve. Patches greater than a certain size are stable and can, in principle, grow by the precipitation of the remaining dissolved receptors from the supersaturated solution. The task of the crosslinking molecules is to form such stable patches. These considerations are based on a qualitative thermodynamic calculation that takes into account the existence of a boundary tension in a patch (in analogy to the surface tension of a droplet). Thermodynamically, these systems should cap spontaneously after the patches have reached a certain size. But, in practice, such a process can be very slow. A suspension of patches may stay practically stable. The ways in which a cell may abolish this metastable equilibrium and thus achieve capping are considered and possible effects of capping inhibitors are discussed.


Thyroid hormones control lysosomal enzyme activities in liver and skeletal muscle. Because protein degradation in liver and skeletal muscle is increased by thyroid hormones and decreased by thyroidectomy; we investigated the influence of thyroid hormones on the level of lysosomal enzymes. Hypophysectomized rats received daily injections of L-thyroxine or L-triiodothyronine. After 3 days of this regimen, homogenates of liver and skeletal muscle showed a 2- to 3-fold increase in the activities of cathepsin D, cathepsin B, and other lysosomal enzymes including leucine aminopeptidase, acid phosphatase, beta-galactosidase, N-acetylglucosaminidase, and alpha-mannosidase. In liver, this effect reflected increased enzyme activity in the two subcellular fractions that normally contain lysosomes. Titration of cathepsin D with pepstatin indicated that the increase in this activity resulted from an increase in the number of enzyme molecules. These effects occurred with both pharmacologic (thyrotoxic) and physiologic (growth-promoting) doses of thyroid hormones. Liver and skeletal muscle from thyroidectomized rats had approximately 50% of the normal levels of lysosomal enzyme activities. Under these various conditions, heart and kidney, tissues in which protein degradation does not appear to be influenced by thyroid hormones, showed no significant changes in lysosomal hydrolases. Thus, thyroid hormones regulate proteolytic and other lysosomal enzyme activities in those tissues in which these hormones influence protein degradation. Many characteristic features of hyperthyroidism and hypothyroidism may result from changes in levels of lysosomal enzymes.