Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences

The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA.     

Mechanisms of spacing in groups of juncos: Measurement of behavioural tendencies in social situations

To investigate behavioural mechanisms that regulate the spacing of individuals, we studied aggression, approach, and avoidance of captive male juncos (Junco hyemalis) in relation to the distance between individuals. We measured behavioural tendencies by analysing the terminations of social states so as to determine the instantaneous probability rates of terminal actions. These rates permit accurate comparisons of behavioural tendencies in different social situations. An individual's tendencies to supplant, approach, or withdraw from an opponent depended on the dominance relationship of the two birds and on the distance between them. Interactions did not vary with the magnitude of the difference between opponents' ranks. The dependence of aggression, approach, and avoidance on the distance between opponents controls the spacing of individuals.     

Induced recovery of a suppressed idiotype by immunization with anti-idiotype

Neonatal Balb/c mice were suppressed forthe H8/T15 idiotype by injection of homologous anti-H8 (D. S. Strayer, D. A. Rowley, and H. Köhler, et al J. Immunol.114, 722, 1975. Six to eight weeks later groups of these suppressed mice were immunized up to three times with isologous anti-H8 raised in Balb/c. After a rest of 4 weeks each group was challenged with R36a vaccine and bleedings were obtained before and after this challenge. All sera were assayed for total anti-PC and H8 idiotype amounts by solid-phase radioimmunoassay. After two-preimmunizations with isologous anti-H8 no increase of the H8 levels was observed though these mice responded to R36a immunization with an increase of total anti-PC antibodies. After the third preimmunization, however, the H8 idiotype was increased in sera taken before and after challenge with R36a. These findings demonstrate that the state of neonatal idiotype suppression can be broken by immunization with complementary anti-idiotype.     

On the application of polyelectrolyte limiting laws to the helix–coil transition of DNA. V. Ionic effects on renaturation kinetics

The bimolecular rate constant k₂ for the association of complementary polynucleotide strands has been observed to increase strongly with increasing ionic strength—in fact, proportional to its third or fourth power. This effect is here interpreted quantitatively by means of polyelectrolyte theory starting with the Wetmur–Davidson postulate of a pre-equilibrium between separated strands and aligned segments close to one another but unbonded. The correct form, a power dependence of k₂ on ionic strength, is predicted. Comparison of the theoretical exponent with data allows the conclusion that each of the two single-stranded segments in the aligned but unbonded configuration consists of about 13–16 nucletides (not to be confused with the much smaller number of bonded base pairs in the nucleus), and that this number, denoted by Q, is possibly correlated either with a minimum length for duplex stability or with the persistence length of a single polynucleotide strand. It is suggested that experimental determination of the dependence of Q on (G+C)-content may distinguish between these possibilities. It is also suggested that addition of sufficient amounts of divalent metal ions such as Mg²⁺, Ca²⁺, or Co²⁺ may reverse the dependence of k₂ on ionic strength; under these conditions, k₂ is predicted to decrease with about the first power of ionic strength. At fixed ionic strength, k₂ should increase with increasing concentration of divalent metal ion, and, in fact, the published observation that the formation of poly(A)·2 poly(U) from poly(A)·poly(U) and poly(U) is second order in Mg²⁺ concentration is here correctly predicted from a priori molecular considerations. Finally, published association rate data for oligonucleotides are discussed in the present theoretical context.     

PINEAL SEROTONIN N-ACETYLTRANSFERASE ACTIVITY: PROTECTION OF STIMULATED ACTIVITY BY ACETYL-CoA AND RELATED COMPOUNDS

—Rat pineal serotonin N—acetyltransferase activity increases 30–70-fold at night in the dark and then decreases rapidly when animals are exposed to light. Activity of the enzyme is also stimulated by l -norepinephrine in organ culture. When homogenates of glands stimulated by dark in vivo or NE in vitro are incubated at 37°C, enzyme activity will also rapidly decrease. This decrease can be prevented by one of the cosubstrates of the enzyme, acetyl–CoA. Protection can also be conferred by cysteamine (β-mercaptoethylamine, HS–CH₂–CH₂–NH₂) which is the terminal portion of the CoA molecule. This protection mechanism could be involved in the physiological control of enzyme activity.     

A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.

The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride.     

Sexual reproduction and parent-offspring conflict in the RNA "Tumour" virus/host relationship: implications for vertebrate oncogene evolution.

Longmuir and co-workers have reported that respiration of certain tissue slices is approximated by Michaelis-Menten kinetics. From this and other experimental findings, Longmuir proposed that a carrier is involved in tissue oxygen transport. Gold developed a deterministic model to examine this hypothesis. This report presents a stochastic model for a fixed site carrier in a more general framework that includes the stochastic counter-part to Gold's deterministic model as a special case. The kinetics of tissue oxygen consumption predicted by the model are examined for various cases.