On the stochastic model for estimation of mutational distance between homologous proteins

Summary A set of simple equations is derived which gives the relationship between the observed amino acid differences per 100 codons and the evolutionary distance per 100 codons using Holmquist's stochastic model of molecular evolution.Contribution No. 910 from the National Institute of Genetics, Mishima, Shizuoka-ken 411 Japan.     

On the rate of molecular evolution

Summary There are at least two outstanding features that characterize the rate of evolution at the molecular level as compared with that at the phenotypic level. They are; (1) remarkable uniformity for each molecule, and (2) very high overall rate when extrapolated to the whole DNA content.The population dynamics for the rate of mutant substitution was developed, and it was shown that if mutant substitutions in the population are carried out mainly by natural selection, the rate of substitution is given byk = 4 N _e s ₁ v, whereN _e is the effective population number,s ₁ is the selective advantage of the mutants, andv is the mutation rate per gamete for such advantageous mutants (assuming that 4N _e s ₁ 1). On the other hand, if the substitutions are mainly carried out by random fixation of selectively neutral or nearly neutral mutants, we havek = v, wherev is the mutation rate per gamete for such mutants.Reasons were presented for the view that evolutionary change of amino acids in proteins has been mainly caused by random fixation of neutral mutants rather than by natural selection.It was concluded that if this view is correct, we should expect that genes of living fossils have undergone almost as many DNA base replacements as the corresponding genes of more rapidly evolving species.Contribution No. 789 from the National Institute of Genetics, Mishima, Shizuokaken 411 Japan. Aided in part by a grant-in-aid from the Ministry of Education, Japan.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Acetazolamide protects against posthypoxic unstable breathing in the C57BL/6J mouse.

Acetazolamide (Acz), a carbonic anhydrase inhibitor, is used to manage periodic breathing associated with altitude and with heart failure. We examined whether Acz would alter posthypoxic ventilatory behavior in the C57BL/6J (B6) mouse model of recurrent central apnea. Experiments were performed with unanesthetized, awake adult male B6 mice (n = 9), ventilatory behavior was measured using flow-through whole body plethysmography. Mice were given an intraperitoneal injection of either vehicle or Acz (40 mg/kg), and 1 h later they were exposed to 1 min of 8% O(2)-balance N(2) (poikilocapnic hypoxia) or 12% O(2)-3% CO(2)-balance N(2) (isocapnic hypoxia) followed by rapid reoxygenation (100% O(2)). Hypercapnic response (8% CO(2)-balance O(2)) was examined in six mice. With Acz, ventilation, including respiratory frequency, tidal volume, and minute ventilation, in room air was significantly higher and hyperoxic hypercapnic ventilatory responsiveness was generally lower compared with vehicle. Poikilocapnic and isocapnic hypoxic ventilatory responsiveness were similar among treatments. One minute after reoxygenation, animals given Acz exhibited posthypoxic frequency decline, a lower coefficient of variability for frequency, and no tendency toward periodic breathing, compared with vehicle treatment. We conclude that Acz improves unstable breathing in the B6 model, without altering hypoxic response or producing short-term potentiation, but with some blunting of hypercapnic responsiveness.     

Physiological Changes of Rhodotorula glutinis Induced by an Acid Protease Inhibitor (S–PI)

An acid protease inhibitor (S–PI) accelerated the growth of Rhodotorula glutinis. This acceleration of the growth was observed only when the pH of the culture fluid lowered during the cultivation, but was not observed when the pH of the culture fluid was adjusted to 2.8 with NaOH. The increase in number of cells was closely related to cell deaths. The cell deaths were observed in an active growing state but not in a resting state. S–PI increased the DNA, RNA and protein content in the cell and decreased the lipid, especially the phospholipid content. When the yeast was cultured in an acidic medium having an initial pH of 1.8 in the presence of S–PI, the decrease of the phospholipid in the cell preceded, followed by the leakage of potassium ion, UV absorbing materials and free pool amino acid from the cell which were accompanied with the cell death. On the basis of these results, physiological changes occuring in the yeast cell are discussed.     

Polygenetic susceptibility and resistance to 4-nitroquinoline 1-oxide-induced tongue carcinomas in the rat

Oral administration of 4-nitroquinoline 1-oxide (4NQO) to rats induced a high incidence of tongue carcinomas (TCs). The inbred Dark-Agouti (DA) strain of rats showed much higher susceptibility to 4NQO-induced TCs than the Wistar-Furth (WF) strain. Our previous study on crosses between the two strains postulated a semidominant susceptibility gene in DA and a semidominant resistance gene in WF rats. This hypothesis was confirmed by the genetic analysis of the back-crosses to either parent with PCR-based microsatellite assay. Using the number of TCS with >5 mm diameter as a quantitative parameter, we mapped a quantitative trait locus Stc1 (Susceptibility to TC) favouring TC development near the locus D19Mit9 on Chr. 19 with a peak LOD score of 6.08. Two other regions in Chr. 3 and Chr. 14 showed weak linkage for susceptibility, but were not statistically significant. On the other hand, another quantitative trait locus Rtc1 (Resistance to TC) providing resistance to TCs was mapped on Chr. 1 between the loci of D1Mit1 and D1Mit3 with a peak LOD score of 3.30. Quantitative parameters such as the number of tumours in the tongue or upper alimentary tract, the frequency of larger tumours and their maximum size were closely correlated and principally determined by Stc1 and Rtc1. Therefore the susceptibility to 4NQO-induced TCs in crosses between DA and WF is explained by the combinations of genotypes at these two loci. Possible candidate genes for Stc1 and Rtc1 are discussed.     

Modification of delayed-type hypersensitivity reactions by muramyldipeptide in guinea pigs

Abstract N -Acetylmuramyl- l -alanyl- d -isoglutamine (muramyldipeptide, MDP) modulated delayed-type hypersensitivity (DTH) reactions and induced severe inflammatory lesions in guinea pigs. The animals immunized with heat-killed Mycobacterium tuberculosis were challenged with the purified protein derivative (PPD) at the flanks and the corneas to prepare DTH reactions at 2 weeks after the immunization, thereafter 24 h the animals received subcutaneous injections of MDP at the flanks of the opposite side. At the skin with the DTH reaction, increase of swelling and redness accompanied with hemorrhage and necrosis were observed. As corneal reactions in the animals that had received MDP, increase of cornea thickness, opaque and grayish-white and the projection of eyes accompanied with severe iritis were observed. Modification of the skin reaction occurred from 2 h after the MDP injection, rapidly increased to the maximum level around 10 h, maintained the level until 24 h, then slowly decreased. The polymorphonuclear leukocyte infiltration was observed from 15 min after the MDP injection, and tumor necrosis factor alpha, interleukin (IL)-1, and IL-6 levels in the serum and skin lesions increased after the MDP injection. Synthetic muramyltripeptide ( N -acetylmuramyl- l -alanyl- d -isoglutaminyl- l -lysine) also provoked definite skin reactions, while the larger peptidoglycan fragments and various inflammatory agents including cytokines so far examined were inactive in this respect. Cortisone and heparin inhibited definitely and slightly the reaction, respectively. A comparison was made with the modified DTH reaction and the necrotic reactions which we reported previously.