Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation.

The amino acid sequences of 25 archaeal retinal proteins from 13 different strains of extreme halophiles were analyzed to establish their molecular phylogenetic relationship. On the basis of amino acid sequence similarity, these proteins apparently formed a distinct family designated as the archaeal rhodopsin family (ARF), which was not related to other known proteins, including G protein-coupled receptors. The archaeal rhodopsin family was further divided into four clusters with different functions; H+ pump (bacteriorhodopsin), Cl- pump (halorhodopsin), and two kinds of sensor (sensory rhodopsin and phoborhodopsin). These four rhodopsin clusters seemed to have occurred by gene duplication(s) before the generic speciation of halophilic archaea, based on phylogenetic analysis. Therefore, the degrees of differences in amino acid sequences within each cluster simply reflected the divergent evolution of halophilic archaea. By comparing the branch lengths after speciation points of the reconstituted tree, we calculated the relative evolution rates of the four archaeal rhodopsins bacteriorhodopsin:halorhodopsin:sensory rhodopsin: phoborhodopsin to be 5:4:3:10. From these values, the degrees of functional and structural restriction of each protein can be inferred. The branching topology of four clusters grouped bacteriorhodopsin and halorhodopsin versus sensory rhodopsin and phoborhodopsin by likelihood mapping. Using bacteriorhodopsin (and halorhodopsin) as an outgroup, the gene duplication point of sensory rhodopsin/phoborhodopsin was determined. By calculating the branch lengths between the gene duplication point and each halophilic archaea speciation point, we could speculate upon the relative evolution rate of pre-sensory rhodopsin and pre-phoborhodopsin. The evolution rate of pre-sensory rhodopsin was fivefold faster than that of pre-phoborhodopsin, which suggests that the original function of the ancestral sensor was similar to that of phoborhodopsin, and that sensory rhodopsin evolved from pre-sensory rhodopsin by the accumulation of mutations. The changes in evolution rate by gene duplication and functional differentiation were demonstrated in the archaeal rhodopsin family using the gene duplication date and halobacterial speciation date as common time stamps.

Identification of proteolipid from an extremely halophilic archaeon Halobacterium salinarum as an N,N'-dicyclohexyl-carbodiimide binding subunit of ATP synthase.

ATP synthesis in an extremely halophilic archaeon, Halobacterium salinarum, was inhibited by N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), a fluorescent analog of N,N'-dicyclohexylcarbodiimide (DCCD). By tracing the fluorescent signal, a hydrophobic 8-kDa protein (proteolipid) was purified from the halobacterial membrane as one of the most DCCD-reactive proteins and its N-terminal amino acid sequence was determined. The gene encoding the proteolipid was found in the region upstream of the genes encoding the two major subunits of halobacterial A-type ATPase [K. Ihara and Y.Mukohata (1991) Arch. Biochem. Biophys. 286, 111-116]. Halobacterial proteolipid was more similar in size to the proteolipid of F-type ATPase than that of V-type ATPase. However, multiple amino acid sequence alignment of proteolipids showed a higher degree of relatedness between V-type and A-type ATPase proteolipids. Together with the recent finding of a triplicate proteolipid encoding gene from the methanogenic archaeon Methanococcus jannaschii [C. J. Bult et al. (1996) Science 273, 1058-1073], proteolipids from archaea seem to have diverse characteristics in comparison with those from eubacteria or from eukaryotes.

[Effect of radiotherapy against advanced carcinoma of the rectum].

In treating advanced carcinoma of the rectum, we have actively used radiotherapy as an adjunct to surgery to enlarge the group of patients indicating surgery and improve the remote results. Of total 423 patients with rectal cancer treated by us in the past 16 years, 202 patients (47.8%) received radiotherapy. Preoperative radiotherapy was performed on 90 of these patients, postoperative radiotherapy on 73 patients, preoperative plus postoperative radiotherapy on 15 patients, and radiotherapy alone on 24 patients. The present study is mainly concerned with preoperative radiotherapy. The purpose of preoperative radiotherapy is to increase the number of patients suitable for surgery. We selected Dukes-C cases without distant metastasis as indications for preoperative radiotherapy, and treated them by 2-step irradiation with 3,000-6,000 rads using Linac after 2 weeks of preoperative observation. The gross and histologic evaluation of the results of irradiation permitted us to conclude that over 3,000 rads was necessary for preoperative radiotherapy. Comparison of the results between preoperative radiotherapy and control groups showed that local recurrence occurred in 6.7% of the radiotherapy group and 9.0% of the control, and 5-year-survival rate was 62.7% and 53.2% respectively, with prognosis improved in the radiotherapy group.