Investigation of the specificity of O6-alkylguanine-DNA-alkyltransferase

Comparison of the abilities of alkylated RNA and DNA to serve as substrates for the O6-alkylguanine-DNA-alkyltransferase have been carried out. It was found that the O6-methylguanine in tRNA was much less active as a substrate for the protein than O6-methylguanine in double stranded DNA. The difference in rates of repair was such that it is unlikely that the alkyltransferase would act on RNA in vivo and, therefore, the reaction with RNA should not contribute towards the exhaustion of its repair capacity.     

A patient with hypogonadotropic hypogonadism successfully treated by long-term pulsatile administration of luteinizing hormone-releasing hormone

A male patient with hypogonadotropic hypogonadism has been treated by pulsatile administration lf luteinizing hormone-releasing hormone (LHRH) (20-25 micrograms, every 2 hours, sc) for 4 years 6 months. His plasma testosterone (T) concentration began to increase after 4 weeks of treatment and reached the normal range in week 5. He showed complete secondary sexual development after 1 year of treatment. His sperm count was normalized after 1 year of treatment. He was married after 29 months of therapy, and has a healthy male child. Blood type tests showed his paternity of the child. During the long duration of pulsatile LHRH therapy, his gonadotropin secretion has been stimulated by LHRH and his T level has been maintained with no observable side effects. There are no other reports of patients treated by pulsatile LHRH injection for such a long duration, but finding in this patient indicated that long-term pulsatile LHRH therapy is a useful and safe method for treatment of hypothalamic hypogonadotropic hypogonadism.     

Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.     

The number of nucleotides required to determine the branching order of three species,with special reference to the human-chimpanzee-gorilla divergence

Summary A mathematical theory for computing the probabilities of various nucleotide configurations among related species is developed, and the probability of obtaining the correct tree (topology) from nucleotide sequence data is evaluated using models of evolutionary trees that are close to the tree of mitochondrial DNAs from human, chimpanzee, gorilla, orangutan, and gibbon. Special attention is given to the number of nucleotides required to resolve the branching order among the three most closely related organisms (human, chimpanzee, and gorilla). If the extent of DNA divergence is close to that obtained by Brown et al. for mitochondrial DNA and if sequence data are available only for the three most closely related organisms, the number of nucleotides (m^*) required to obtain the correct tree with a probability of 95% is about 4700. If sequence data for two outgroup species (orangutan and gibbon) are available, m^* becomes about 2600–2700 when the transformed distance, distance-Wagner, maximum parsimony, or compatibility method is used. In the unweighted pair-group method, m^* is not affected by the availability of data from outgroup species. When these five different tree-making methods, as well as Fitch and Margoliash's method, are applied to the mitochondrial DNA data (1834 bp) obtained by Brown et al. and by Hixson and Brown, they all give the same phylogenetic tree, in which human and chimpanzee are most closely related. However, the trees considered here are gene trees, and to obtain the correct species tree, sequence data for several independent loci must be used.     

Fever in rats during normal and dehydrated conditions

The effect of endogenous pyrogen (EP, from rabbit) and endotoxin (Salmonella typhosa) on rectal temperature (Tre) was investigated in normal and dehydrated rats of both sexes. Intraperitoneal injection of either EP or endotoxin did not affect body temperature. In addition, no changes in Tre were observed when endotoxin was injected intravenously in normally hydrated male rats, but significant falls in Tre occurred in normal female rats. However, intravenous injection of EP produced fever in both sexes, but females generally showed smaller responses. A second intravenous injection of endotoxin, given 3 days after the first injection, always produced fever in normally hydrated rats. The pattern of this febrile response was monophasic. In contrast to the response in normal rats, intravenous endotoxin produced significant fevers with a biphasic pattern in dehydrated rats of either sex, but the febrile responses of male rats were greater than those of female rats. On the other hand, there were no significant differences between febrile responses to intravenous EP exhibited by normal and dehydrated animals. These results show that rats of both sexes possess physiological mechanisms capable of producing a fever following intravenous injections of EP.     

Gene Genealogy and Variance of Interpopulational Nucleotide Differences

A mathematical theory is developed for computing the probability that m genes sampled from one population (species) and n genes sampled from another are derived from l genes that existed at the time of population splitting. The expected time of divergence between the two most closely related genes sampled from two different populations and the time of divergence (coalescence) of all genes sampled are studied by using this theory. It is shown that the time of divergence between the two most closely related genes can be used as an approximate estimate of the time of population splitting (T) only when T identical to t/(2N) is small, where t and N are the number of generations and the effective population size, respectively. The variance of Nei and Li's estimate (d) of the number of net nucleotide differences between two populations is also studied. It is shown that the standard error (Sd) of d is larger than the mean when T is small (T much less than 1). In this case, Sd is reduced considerably by increasing sample size. When T is large (T greater than 1), however, a large proportion of the variance of d is caused by stochastic factors, and increase in the sample size does not help to reduce Sd. To reduce the stochastic variance of d, one must use data from many independent unlinked gene loci.     

Models of Evolution of Reproductive Isolation

Mathematical models are presented for the evolution of postmating and premating reproductive isolation. In the case of postmating isolation it is assumed that hybrid sterility or inviability is caused by incompatibility of alleles at one or two loci, and evolution of reproductive isolation occurs by random fixation of different incompatibility alleles in different populations. Mutations are assumed to occur following either the stepwise mutation model or the infinite-allele model. Computer simulations by using It?'s stochastic differential equations have shown that in the model used the reproductive isolation mechanism evolves faster in small populations than in large populations when the mutation rate remains the same. In populations of a given size it evolves faster when the number of loci involved is large than when this is small. In general, however, evolution of isolation mechanisms is a very slow process, and it would take thousands to millions of generations if the mutation rate is of the order of 10(-5) per generation. Since gene substitution occurs as a stochastic process, the time required for the establishment of reproductive isolation has a large variance. Although the average time of evolution of isolation mechanisms is very long, substitution of incompatibility genes in a population occurs rather quickly once it starts. The intrapopulational fertility or viability is always very high. In the model of premating isolation it is assumed that mating preference or compatibility is determined by male- and female-limited characters, each of which is controlled by a single locus with multiple alleles, and mating occurs only when the male and female characters are compatible with each other. Computer simulations have shown that the dynamics of evolution of premating isolation mechanism is very similar to that of postmating isolation mechanism, and the mean and variance of the time required for establishment of premating isolation are very large. Theoretical predictions obtained from the present study about the speed of evolution of reproductive isolation are consistent with empirical data available from vertebrate organisms.