An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes.

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

Engineering of Escherichia coli ß-lactamase TEM-1 variants showing higher activity under acidic conditions than at the neutral pH.

Escherichia coli ß-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern. In the present study, we engineered TEM-1 variants preferentially active at pH 5.8-6.2, near the pH of the acidic microenvironment of tumor. A library of randomly mutagenized variants was screened for the ability to confer an antibiotic resistance on E. coli cells in acidic growth media and not in neutral media, to isolate a variant with a Thr-to-Ile substitution at position 160. An extensive mutagenesis study was then conducted in the proximity of this position, to show that a Leu162Glu mutation also causes the acid preference. Kinetic analyses indicated that the overall activity of the wild-type TEM-1 hardly changes over a pH range from 5.8 to 7.0, whereas TEM-1(T160I) is 1.5-times as active at pH 6.2 than pH 7.0, and TEM-1(T160I) is 3.1-fold as active at pH 5.8 than pH 7.0. A further mutagenesis study suggested that a change in the overall structure of the enzyme underlies the pH dependency of the variants.

Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon Reassignments.

In the present study, we simultaneously incorporated two types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense to enhance the utility of this industrial enzyme. The first amino acid, 3-chloro-l-tyrosine, was incorporated into MTG in response to in-frame UAG codons to substitute for the 15 tyrosine residues separately. The two substitutions at positions 20 and 62 were found to each increase thermostability of the enzyme, while the seven substitutions at positions 24, 34, 75, 146, 171, 217, and 310 exhibited neutral effects. Then, these two stabilizing chlorinations were combined with one of the neutral ones, and the most stabilized variant was found to contain 3-chlorotyrosines at positions 20, 62, and 171, exhibiting a half-life 5.1-fold longer than that of the wild-type enzyme at 60 °C. Next, this MTG variant was further modified by incorporating the α-hydroxy acid analogue of Nε-allyloxycarbonyl-l-lysine (AlocKOH), specified by the AGG codon, at the end of the N-terminal inhibitory peptide. We used an Escherichia coli strain previously engineered to have a synthetic genetic code with two codon reassignments for synthesizing MTG variants containing both 3-chlorotyrosine and AlocKOH. The ester bond, thus incorporated into the main chain, efficiently self-cleaved under alkaline conditions (pH 11.0), achieving the autonomous maturation of the thermostabilized MTG. The results suggested that synthetic genetic codes with multiple codon reassignments would be useful for developing the novel designs of enzymes.

Non-machinery dialysis that achieves blood purification therapy without using full-scale dialysis machines.

An electrical or water supply and a blood purification machine are required for renal replacement therapy. There is a possibility that acute kidney injury can occur in large numbers and on a wide scale in the case of a massive earthquake, and there is the potential risk that the current supply will be unable to cope with acute kidney injury cases. However, non-machinery dialysis requires exclusive circuits and has the characteristic of not requiring the full-scale dialysis machines. We performed perfusion experiments that used non-machinery dialysis and recent blood purification machines in 30-min intervals, and the effectiveness of non-machinery dialysis was evaluated by the assessing the removal efficiency of potassium, which causes lethal arrhythmia during acute kidney injury. The non-machinery dialysis potassium removal rate was at the same level as continuous blood purification machines with a dialysate flow rate of 5 L/h after 15 min and continuous blood purification machines with a dialysate flow rate of 3 L/h after 30 min. Non-machinery dialysis required an exclusive dialysate circuit, the frequent need to replace bags, and new dialysate exchanged once every 30 min. However, it can be seen as an effective renal replacement therapy for crush-related acute kidney injury patients, even in locations or facilities not having the full-scale dialysis machines.

The distinctive characteristics of the hourly distribution of live births on specific days in Japan.

OBJECTIVES: Anomalous variations in live births on February 29/March 1, April 1/April 2 and the days before the New Year holidays/New Year holidays have been reported in Japan. The distribution of live births was investigated on those days and whether or not such dates were selected due to obstetric intervention is discussed. METHODS: Using a method similar to the [Formula: see text]-R control chart, anomalous variations in the hourly number of live births were detected. The number of unusual births was estimated. RESULTS: The number of live births at 13:00-16:59 hours was significantly higher from December 24 to 28 and significantly lower from December 29-January 3, February 29, and April 1, especially on weekdays. In hospitals, the increases from 9:00-12:59 and 13:00-16:59 hours from December 24 to 27 were approximately 10 and 25 %, respectively, of the expected births for those times in the mid-1980s; thereafter, the rates were 30 and 35 %. After 2000, the child births at 13:00-16:59 hours on February 29 and April 1 decreased by approximately 35 % in hospitals and clinics. The numbers of live births at 0:00-0:59 hours were significantly higher on March 1 and April 2 until 2001. CONCLUSION: Anomalous variations at 0:00-0:59 hours may be associated with fictitious reporting on birth certificates. Anomalous variations from 13:00 to 16:59 hours on weekdays suggest that many individuals may avoid obstetric intervention on February 29 and April 1 and that the number of higher-risk deliveries may significantly increase in the daytime on the days before the New Year holidays due to obstetric intervention for institutional reasons.

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.

Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25-42°C, and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

Multiple site-specific installations of Nε-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis.

Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.

[The effect of determining birthdays by social factors such as maternal preference on the distribution in the number of daily live births].

OBJECTIVES: To investigate the effect of determining birthdays by social factors on the distribution of daily live births. METHODS: We obtained data on live births tabulated by date and birthplace (hospitals, clinics, and maternity homes) between 1981 and 2010 in Japan from the National Vital Statistics System. This study analyzed the variations in live births for each day of the week, as well as for several specific days observed to have a systematic variation in the number of live births. We determined the ratio of the mean daily live births on those specific days to the overall daily average each month (the birth number ratio). The standardized deviation (the ratio of the deviation to the standard deviation for the day of week) regarding the birth number ratio of each specific day was also determined. RESULTS: The birth number ratio in hospitals and clinics was highest on Tuesdays and lowest on Sundays. Hospitals showed a large difference in the birth number ratio between weekdays and weekends, although the difference in the birth number ratio between weekdays and Saturdays was smaller in clinics than in hospitals. The birth number ratio during the first three days of the New Year was lower than that on Sundays. Until approximately 1995, the standardized deviation on February 29th and April 1st (the end of the Japanese school year) showed abnormally low values, while those on March 1st and April 2nd showed significantly high values. Following that time period, the significant variations on February 29th (only on Sundays), April 1st (only on Sundays), March 1st, and April 2nd almost completely disappeared. Maternity homes showed equivalent results until the 1980s or the middle of the 1990s. CONCLUSION: The variations in the days of the week were inconsistent with nationwide policies for consultation in each setting. These results indicate that some birth dates were set for institutional reasons or maternal preferences (i.e., the day after the leap day or the next school year) by using or avoiding obstetric intervention. The abnormal variation on leap days and on April 1st might be related to fictitious reporting. More recent variations in the birth number ratio on specific days suggest that some individuals may avoid obstetric intervention. The results of this study indicate that determining birthdays by social factors may have been practiced in maternity homes until approximately 1990.

[Characteristics of infection prevention and coping behavior for seasonal influenza-like illnesses and its relationship to personal characteristics among hospital nurses].

OBJECTIVES: To describe the infection prevention and coping behavior for seasonal influenza-like illnesses among hospital nurses. METHODS: We conducted an anonymous questionnaire survey of 444 nurses in October 2007, who belonged to two hospitals in one city. We investigated their infection prevention behavior (handwashing, gargling, mask-use, influenza vaccination rate, humidification of the room, room ventilation, increased physical strength) and coping behavior (type of coping, elapsed time until taking appropriate action, absent days, recognition of infection source) in one season, and their characteristics (sex, age, division, family). RESULTS: 423 questionnaires were analyzed. Most nurses performed handwashing with soap or a disinfectant. However, only 71% and 53% of nurses regularly did this after blowing their nose or touching any hair. Many used only water. Only 58% of the nurses gargled at home. Except after handling linen, gargling was done by less than 10%. Regarding handwashing or gargling, nurses who performed these before the beginning of duties or any treatment was only in the range from 10-25% which was less than when they finished their duties or treatment. Handwashing before beginning duties was significantly associated with "living together with a family" (odds ratio [95% confidence interval] after adjusting for sex and age) (0.32[0.12-0.84]) and "living together with children who go to school" (0.49[0.24-0.995]), respectively. Gargling after any treatment and gargling at home, room humidification and ventilation were all significantly associated with "living together with babies and infants" (2.36[1.07-5.21], 1.87[1.07-3.27], 2.29[1.32-3.97] and 2.46[1.39-4.36]). Fifty-five% of the nurses regularly wore masks during work. The influenza vaccination rate was 82%. 67% of 51 nurses who had flu-like symptoms responded appropriately within 24 hours after onset. However, 25% of 51 nurses did not consult a doctor, but instead took over-the-counter medicine or rested at home. Some 28% of 51 nurses did not miss any work days, many not wanting to take time off due to insufficient personnel. 22% of the nurses thus were found to become an infection source. At one hospital, it was more necessary to improve coping behavior, while at another hospital, it was more necessary to improve "handwashing before setting the table" and "gargling after handling linen". CONCLUSION: These findings suggest that the infection prevention awareness toward patients was lower than self-infection prevention awareness of nurses and that these were related to such members living together as a family and their place of work. Gargling was not sufficiently performed. These results suggest a need to educate nurses about "coughing etiquette", "handwashing before contacting patients", and "gargling at work and home", and to establish an effective medical protocol where the nurses can consult a medical specialist when they suspect they may have an infection themselves.

Structural basis of abscisic acid signalling.

The phytohormone abscisic acid (ABA) mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Within plants, the PYR/PYL/RCAR family of START proteins receives ABA to inhibit the phosphatase activity of the group-A protein phosphatases 2C (PP2Cs), which are major negative regulators in ABA signalling. Here we present the crystal structures of the ABA receptor PYL1 bound with (+)-ABA, and the complex formed by the further binding of (+)-ABA-bound PYL1 with the PP2C protein ABI1. PYL1 binds (+)-ABA using the START-protein-specific ligand-binding site, thereby forming a hydrophobic pocket on the surface of the closed lid. (+)-ABA-bound PYL1 tightly interacts with a PP2C domain of ABI1 by using the hydrophobic pocket to cover the active site of ABI1 like a plug. Our results reveal the structural basis of the mechanism of (+)-ABA-dependent inhibition of ABI1 by PYL1 in ABA signalling.

[Estimation of excess mortality associated with influenza epidemics specific for sex, age and cause of death in Japan during 1987-2005].

OBJECTIVES: The aim of this study is to clarify the excess mortality associated with influenza epidemics in Japan during the period from 1987 to 2005. METHODS: Monthly data on the total number of deaths (excluding accidental deaths) and the numbers of deaths due to malignant neoplasms, heart disease, cerebrovascular disease, pneumonia, and renal failure were obtained from vital statistics from 1987-2005. The point estimates and range of excess mortality were evaluated using a model based on annual mortality and seasonal indices. Total mortality was analyzed for all ages, sex and for the following five age groups: 0-4, 5-24, 25-44, 45-64, and > or =65 yrs. RESULTS: The excess number of deaths showed almost no difference in each influenza season between men and women. During each influenza season, approximately 85-90% of the excess mortality was attributed to the > or =65 yrs age group. During the 1995 and 1999 seasons, mortality increased significantly across all age groups. The highest point estimate of excess mortality in the > or =65 yrs age group was observed in 1999. From a comparison of the range of excess mortality in the > or =65 yrs age group by year, the excess mortality in 1995 appeared to be the highest of the examined years. The highest point estimate of excess mortality in the 0-4 yrs age group was observed in 1995. From a comparison of the range of excess mortality in the 0-4 yrs age group by year, the excess mortality in 1998 or 1999 appeared to be the highest of the examined years. Excess mortality in the 45-64 yrs and > or =65 yrs age groups showed an increasing tendency in the 1990s and a stabilizing tendency beginning in 2000. In addition, excess mortality during each epidemic was occurred in persons with pneumonia, heart disease, cerebrovascular disease, malignant neoplasms, and renal failure, accounting for approximately 20-50%, 20-40%, 20%, 5%, and 2% of all the excess mortality, respectively. CONCLUSIONS: These results indicate that the majority of excess mortality occurred among the elderly and persons with pneumonia, heart disease, or cerebrovascular disease. Although it is unclear whether the increasing trend in the 1990s and the stabilizing trend beginning in 2000 were the result of vaccination measures, health measures for groups such as the elderly and heart disease patients are considered to be important for the future.

Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal-cell morphogenesis in Arabidopsis.

The involvement of kinesin motor proteins in both cell-tip growth and cell-shape determination has been well characterized in various organisms. However, the functions of kinesins during cell morphogenesis in higher plants remain largely unknown. In the current study, we demonstrate that an armadillo repeat-containing kinesin-related protein, ARMADILLO REPEAT KINESIN1 (ARK1), is involved in root-hair morphogenesis. Microtubule polymers are more abundant in ark1 null allele root hairs, but analysis shows that these extra microtubules are concentrated in the endoplasm, and not in the cortical array, suggesting that ARK1 regulates tip growth by limiting the assembly and distribution of endoplasmic microtubules. The ARK1 gene has two homologues in the Arabidopsis genome, ARK2 and ARK3, and our results show that ARK2 is involved in root-cell morphogenesis. We further reveal that a NIMA-related protein kinase, NEK6, binds to the ARK family proteins and has pleiotropic effects on epidermal-cell morphogenesis, suggesting that NEK6 is involved in cell morphogenesis in Arabidopsis via microtubule functions associated with these armadillo repeat-containing kinesins. We discuss the function of NIMA-related protein kinases and armadillo repeat-containing kinesins in the cell morphogenesis of eukaryotes.

Centrin controls the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels Ca2+-dependently.

In Paramecium, ciliary reversal is coupled with voltage-gated Ca(2+) channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca(2+) channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca(2+)-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca(2+) channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca(2+) sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca(2+)-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca(2+)-dependently regulates the Ca(2+) channel activity in vivo.

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors.

Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is an ATP-driven Ca(2+) pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca(2+), the three cytoplasmic domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca(2+)-ATPase in the absence of Ca(2+) stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1-M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1' helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca(2+)-ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations.

[Estimation of the range of excess death associated with influenza epidemics: application of a model using annual mortality rates and the seasonal index for determining minimum excess mortality estimates].

OBJECTIVE: To determine the range of excess death associated with influenza in Japan. METHODS: The monthly rates for mortality from all causes other than accidents (ICD9: E800-E949, ICD-10: V01-X59) were determined from the Japanese national vital statistics for 1987-2003. By employing a model using the annual mortality rates and the seasonal index, the number of expected deaths and the 95% range of mortality in the absence of influenza-associated deaths (normal range) were obtained. Point estimation of excess mortality during an influenza season was performed by calculation of differences between the number of deaths observed and the number of deaths expected. The range of excess death was defined: the lower limit of the excess death was performed by calculation of difference between the number of deaths observed and the upper limit of the normal range. The upper limit of the excess death, on the contrary, was performed by calculation of difference between the number of deaths observed and the lower limit of the normal range. Based on the results of a survey of tendencies in the occurrence of infectious diseases, months showing a high prevalence of influenza and associated deaths were regarded as "months when the rate of death from influenza was 0.9 deaths/100,000 person-years or higher". RESULTS: The excess death determined by point estimation was largest in 1999, followed by 1995, 1993, 1997, 2000, and 2003. The point estimation (range of excess death) in 1999 was approximately 49,000 (approximate range, 37,000-60,000). Correspondingly, the excess death in 1995 was approximately 38,000 (approximate range, 27,000-48,000). While values in 1993, 1997, 2000, and 2003 were in the approximate range of 21,000 to 25,000, the range of excess death of each year showed the differences: approximate range from 15,000 to 36,000 in 1993, from 18,000 to 31,000 in 1997, from 18,000 to 28,000 in 2000, and from 11,000 to 34,000 in 2003, respectively. From comparison of the range of excess deaths by year, that in 1995 appeared to be the largest among the examined years. CONCLUSION: By considering the range of variation in the number of deaths in the years when no influenza epidemics occurred, the increase (the largest number and smallest number of deaths) associated with elevation the prevalence of influenza could be determined. Estimation of the range of excess deaths can be considered a useful method for understanding the influence of influenza on public health.

Non-Mendelian inheritance induced by gene amplification in the germ nucleus of Paramecium tetraurelia.

A genetic investigation of strain d4-95, which carries a recessive mutant allele (pwB(95)) of pawn-B, one of the controlling elements of voltage-dependent calcium channels in Paramecium tetraurelia, revealed a non-Mendelian feature. Progeny of the cross between d4-95 and wild type often expressed a clonally stable mutant phenotype, even when they had a wild-type gene. The mutant phenotype was also expressed after self-fertilization of theoretical wild-type homozygotes recovered from the cross. Our molecular analysis demonstrated that the copy number of the mutant pwB gene in the micro- and macronucleus of d4-95 was much greater than that of the wild type. Most of the amplified, extra pwB gene copies in d4-95 were heritable independently from the original pwB locus. Repeated backcrossing of d4-95 with the wild type to dilute extra pwB genes in the strain produced segregants with a completely normal Mendelian trait in testcrosses. These results strongly suggest that a non-Mendelian inheritance of d4-95 was induced by gene amplification in the micronucleus.

Membrane potential responses of paramecium caudatum to external Na+.

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.