Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila.

The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about 1 h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2 h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB, more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca2+ channels.     

Intra- and interspecific complementation of membrane-inexcitable mutants of Paramecium

Membrane excitation was the basis for backward swimming of Paramecium facing stimulus. According to standard genetic tests, inexcitable mutants fell into three complementation groups for both Paramecium tetraurelia (pwA, pwB, and pwC) and Paramecium caudatum (cnrA, cnrB, and cnrC). Cytoplasm from a wild type transferred to a mutant through microinjection restored the excitability. Transfusions between genetically defined complementation groups of the same species effected curing, whereas transfusions between different mutants (alleles) of the same group or between sister cells of the same mutant clone did not. Cytoplasmic transfers of all combinations among the six groups of mutants of the two species showed that any cytoplasm, except those from the same group, was able to cure. Since the pawns and the caudatum nonreversals complement one another through transfusion, they appeared to belong to six different complementation groups. The extent of curing, the amount of transfer needed to cure, and the time course of curing were characteristic of the group that received the transfusion. Variations in these parameters further suggested that the six groups represented six different genes. Because the donor cytoplasms from either species were equally effective quantitatively in curing a given mutant, the curing factors were not species specific. These factors are discussed.     

Behavioral Mutants in PARAMECIUM CAUDATUM

Mutants of Paramecium caudatum with abnormal swimming behavior or responses to cations were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Some of the mutants, like pawn in P. tetraurelia, cannot swim backward and are called CNR. Seven independently obtained CNR mutants belonged to three complementation groups, designated as cnrA, cnrB and cnrC. Some characteristics of double homo- and heterozygotes were compared with single homo- and heterozygotes. Other behavioral mutants shown to have a genic basis included K⁺-sensitive, temperature-shock behavioral and slow swimmer. All those mutants except for slow swimmer had lesions in the membrane because Triton-extracted models of them show almost the same swimming behavior as wild type.     

Characterization and purification of a soluble protein controlling Ca-channel activity in paramecium

The analysis of the voltage-sensitive Ca++ channel of the unicellular eucaryote, Paramecium has been extended to a biochemical level based on recent observations that the transfer of cytoplasm from wild-type cells into mutants lacking Ca++-channel function ("pawn" in P. tetraurelia and "CNR" in P. caudatum) causes mutant cells to regain Ca++-channel function. We have microinjected various cytoplasmic fractions into mutant cells and measured the restored Ca++-channel function using a convenient behavioral assay. Following the "curing" activity, we characterized and purified the component from wild-type cytoplasm that can restore the function missing in cells carrying mutations in the cnrC gene. The curing factor is not an RNA, but a heat-labile, -SH-containing protein that appears to affect existing mutant channels on the ciliary membrane. We have purified this factor over 500-fold from the soluble cytoplasm using conventional techniques. The protein is of low apparent molecular weight (less than 30,000 daltons), acidic, soluble, and does not have the properties of calmodulin.     

Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila

The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about l h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca²⁺ channels. © 1992 Wiley-Liss, Inc.     

Roles of sexual cell agglutination in yeast mass mating

The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia.     

Biochemical and cellular changes occuring during conjugation in Hansenula wingei

Brock, Thomas D. (Indiana University, Bloomington). Biochemical and cellular changes occurring during conjugation in Hansenula wingei. J. Bacteriol. 90:1019-1025. 1954.-A technique has been devised for deagglutinating mixed populations of conjugating cells so as to be able to visualize microscopically early stages of the conjugation process. A cell can form a conjugation tube only when in contact with a cell of opposite mating type, but may do so even if the mate is unresponsive or ultraviolet-inactivated. Cell fusion occurs, however, only when both cells are able to form conjugation tubes in a region of contact. Fusion begins almost as soon as the two cells begin to form protuberances, and long before any dissolution of cell-wall material between the cells occurs. A cell which has conjugated in one region of its cell wall is still able to conjugate with another cell in another region, so that triply and quadruply conjugated cells are occasionally formed. There is no significant net increase in deoxyribonucleic acid, ribonucleic acid, protein, or carbohydrate which might be related to the conjugation process, because any minor changes that occur in these components are also detected when cells of only one mating type are incubated or when the conjugation process is inhibited with the antibiotic cycloheximide. Changes in activity of beta-1,3-glucanase (with laminarin as substrate) and beta-1,6-glucanase (with pustulan as substrate) have been measured during the conjugation process, in addition to changes in the activity of several control enzymes which would not be expected to be related to the conjugation process. Significant increases in invertase (sucrase), laminarinase, and pustulanase were detected, and minimal increases occurred in beta-glucosidase and acid phosphatase. However, these same increases were also observed in controls involving only one mating type; thus, these increases are probably not related to the conjugation process, but may be a result of other processes which probably occur during incubation in the conjugation medium.     

Kin selection is implicated in partial sib-mating populations with constant viability differences before mating

The change in the frequency of a rare mutant allele under constant sex-differentiated viability selection in an infinite, partial full-sib mating population is studied. The diplo-diploid and haplo-diploid polygynous models are considered with a Poisson distribution for the number of offspring produced by every mated female. Reproduction is followed by weak selection among the offspring and then mating to form the next generation. It is shown that the rate of change with respect to the frequency of the mutant allele and the intensity of selection can be expressed in terms of costs or benefits of substituting the mutant type for the wild type, which correspond to average excesses in viability in females and males, multiplied by coefficients of relatedness to the individuals affected by such a substitution and reproductive values associated to the sexes of these individuals. This reveals hidden interactions between mated individuals and between males for mating, the former having positive effects on the reproductive success of related individuals and the latter having negative effects. Such interactions are the result of reproductive constraints when a fixed proportion of females must mate with a male sib and all females are fertilized as long as one mate is available. However, they affect the change in allele frequency because there is inbreeding or relatedness between mates and more generally relatedness between interacting individuals. Surprisingly, the effects of these interactions cancel out in a diploid population when the number of offspring is large enough so that the possibility for a female to have no male sib to mate with can be neglected and the viability differences are the same in both sexes.     

Ultrastructural study of the life cycle of <Emphasis Type="Italic">Rickettsia slovaca</Emphasis>, wild and standard type,cultivated in L929 and vero cell lines

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.     

Phenotypic accommodation: adaptive innovation due to developmental plasticity

Phenotypic accommodation is adaptive adjustment, without genetic change, of variable aspects of the phenotype following a novel input during development. Phenotypic accommodation can facilitate the evolution of novel morphology by alleviating the negative effects of change, and by giving a head start to adaptive evolution in a new direction. Whether induced by a mutation or a novel environmental factor, innovative morphological form comes from ancestral developmental responses, not from the novel inducing factor itself. Phenotypic accommodation is the result of adaptive developmental responses, so the novel morphologies that result are not "random" variants, but to some degree reflect past functionality. Phenotypic accommodation is the first step in a process of Darwinian adaptive evolution, or evolution by natural selection, where fitness differences among genetically variable developmental variants cause phenotype-frequency change due to gene-frequency change.     

去极化时Ca~(2+) 内流引起蛋白激酶C_α浆膜转位

 为观察胞外Ca2 + 内流和肌浆网Ca2 + 释放两种来源的Ca2 + 对cPKCα转位激活的影响 ,揭示PKC在去极化 nAChR转录偶联中的作用 ,构建了pPKCα EGFP N1融合蛋白真核基因表达载体 .转染C2C1 2肌细胞后 ,采用激光共聚焦显微镜记录了KC1或咖啡因处理所引起的细胞Ca2 + 波变化及PKCα GFP融合蛋白在细胞内的分布 .结果提示 ,只有用KC1处理引起细胞膜去极化时 ,伴随Ca2 +内流 ,才能观察到PKCα GFP绿色荧光在细胞内发生的细胞浆至细胞膜分布变化 .然而 ,采用肌浆网Ca2 + 通道激动剂咖啡因刺激肌细胞 ,使肌浆网中Ca2 + 释放 ,未见PKCα GFP绿色荧光在浆、膜分布发生任何变化 .结果提示 ,去极化时外Ca2 + 内流可引起PKCα转位激活 ,肌浆网Ca2 + 释放对PKCα的转位激活没有影响 .     

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.     

Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms.

Homozygous c-ros knockout male mice that lack prepubertal differentiation of the epididymal initial segment are healthy but sterile, despite normal sperm production and mating. Detailed computerized analysis of the motility of spermatozoa maturing in the epididymis revealed only minor defects. However, the majority of motile mature sperm released from the cauda epididymidis showed various extents of flagellar angulation that could not be corrected by raising extracellular osmolality. Measurement of the osmolality of cauda epididymal fluid showed no difference from the wild type. Studies in wild-type mice indicated a maturational change in the ability of motile sperm to maintain straight flagella during incubation, but angulation was induced in cauda sperm by the volume-sensitive ion channel blockers quinine, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and BaCl(2), or by exposure to hypotonic media. Flagellar angulation, induced in the wild type or intrinsic to the knockout, was relieved upon demembranation by Triton X-100, confirming that it was a cell swelling phenomenon. A lack of response of immature wild-type sperm and mature knockout sperm to the channel blockers suggests that there is normally a development of the volume regulatory mechanisms upon maturation that is defective in sperm from the knockout animal. The resultant flagellar angulation may account for the reduction in sperm numbers in the oviduct of mated females and the failure to fertilize in vivo.     

Demonstration of Multiple Mating in Heterodera glycines with Biochemical Markers

Controlled crosses of Heterodera glycines were carried out by placing one o r more virgin females of known esterase phenotype on an agar plate and adding, at various time intervals, one or more males of different esterase phenotypes. Progeny (second-stage juveniles) of such crosses were propagated on soybeans, and 30 days later young females were subjected to electrophoretic analysis to determine their esterase phenotype. Esterase phenotypes that represented the heterozygous state of the maternal and paternal genomes confirmed the hybrid nature of the progeny and identified their male parent. When each of 74 females was given the opportunity to mate successively with two males of different esterase phenotypes, 43 mated with a single male and 31 mated with both males. One female mated with three males, i.e., with a male of its own population (sib mating) and the two males provided for the cross. Inseminated females could mate for a second time soon after, or as late as 24 hours after, their first mating. When single males were given the opportunity to mate with many females, about equal numbers of them inseminated zero, one, two, or three females. In greenhouse tests, 12 females were given the opportunity to mate with many males of three different esterase phenotypes. Two females mated with one and possibly more males of the same phenotype, and 10 females mated with males of two different esterase phenotypes. In conclusion, multiple mating appears to be a common behavior of males and females of H. glycines.     

Use of the MHC for mate choice in wild house mice (Mus domesticus)

The mechanisms maintaining natural diversity at the major histocompatibility complex (MHC) are not well understood. To increase knowledge of one potential mechanism, I examined the use of MHC genes for mate choice by wild house mice in a controlled laboratory setting. Three rearing groups of wild test mice were produced: non‐fostered control mice, mice fostered into families of an inbred laboratory mouse strain, and mice fostered into families of a second, MHC‐congenic mouse strain. Mature test mice were given a choice of two opposite‐sex stimulus mice from the two MHC‐congenic strains used for fostering, and were scored for several measures of preference. The results were non‐significant in general, but females of two rearing groups spent significantly more time with mice of one MHC‐type, and in most rearing groups, mice tended to spend more time with this same MHC‐type. Other results showed that male test mice ejaculated indiscriminantly and that female wild mice mated to ejaculation more often in longer length trials, but showed no significant preferences. In this study, fostering seemed to have little or no effect on MHC‐based mate preferences of wild house mice, and wild mice did not appear to be using the MHC to avoid inbreeding. However, some wild female mice used the MHC to choose potential mates. This revised version was published online in July 2006 with corrections to the Cover Date.     

MYO1, a novel, unconventional myosin gene affects endocytosis and macronuclear elongation in Tetrahymena thermophila

Targeted gene disruption was used to investigate the function of MYO1, an unconventional myosin gene in Tetrahymena thermophila. Phenotypic analysis of a transformed strain that lacked a functional MYO1 gene was conducted at both 20 degrees C and 35 degrees C. At either temperature the delta MYO1 strain had a smaller cytoplasm/nucleus ratio than wild type. At 20 degrees C, delta MYO1 populations had a longer doubling time than wild type, lower saturation density, and a reduced rate of food vacuole formation. However, at 35 degrees C, these characteristics were comparable to wild type. Although micronuclear division and cytokinesis appeared normal in delta MYO1 cells, failure of the macronucleus to elongate properly resulted in unequal segregation of macronuclear DNA in cells maintained at either 20 degrees C or 35 degrees C.     

促凋亡蛋白Bid诱导肝细胞凋亡的机制

为研究促凋亡蛋白Bid对肝细胞凋亡过程中的调节机制 ,在体内和体外分别用TNF α或抗Fas抗体诱导小鼠肝细胞凋亡 .免疫荧光染色观察Bax转位和构象变化 ;采用ELISA检测caspase 3和 8的活性 ;Western印迹测定Bid和Bax的裂解活化及Bax的转位和插入 .结果显示 :TNF α或抗Fas抗体通过激活Bid导致Bax转位和构象变化 ,使Bax得以插入线粒体膜诱导肝细胞凋亡 .阻断Bid的作用 ,则Bax的转位和插入明显被削弱 ,肝细胞的凋亡受到抑制 .提示由死亡受体诱导的肝细胞调亡可能受Bid调节 ,Bax转位和插入依赖于Bid .     

The major histocompatibility complex and mating preferences in wild house mice (mus domesticus)

This study examined the relationship between the major histocompatibilitycomplex (MHC) genes and mate choice by wild house mice in acontrolled laboratory setting in an attempt to understand themechanisms maintaining natural MHC diversity. Three rearinggroups of wild test mice were produced: nonfostered controlmice, mice fostered into families of an inbred laboratory mousestrain, and mice fostered into families of a second mouse straindiffering genetically from the first only within the MHC region.At maturity, test mice were given a choice of two opposite-sexstimulus mice of the two MHC-congenic strains used for fostering.Test mice were scored for several measures of preference includingamount of time spent with either stimulus mouse, and ejaculationwith a stimulus mouse. Females in two of three rearing groupsspent more time with one MHC type regardless of rearing environment,suggesting that females did not prefer mates dissimilar fromfamily MHC type. Time preferences tended to be stronger in femalesthan in males. Male test mice ejaculated indiscriminantly. Femalewild mice mated to ejaculation more often in longer trials,but these matings were still too infrequent to assess preferences.Fostering had little or no effect on MHC-based mate preferencesof wild house mice, and no evidence suggested that MHC was usedto avoid inbreeding. Wild female mice may still choose matesbased on MHC haplotypes (but do not necessarily prefer MHC-dissimilarmates); other cues are probably also used. Based on these results,inbreeding avoidance does not seem a strong mechanism for maintainingnatural MHC diversity     

The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.     

The Cross-Linking Agent Hexamethylphosphoramide Predominantly Induces Intra-Locus and Multi-Locus Deletions in Postmeiotic Germ Cells of Drosophila

The nature of DNA sequence changes induced by the cross-linking agent hexamethylphosphoramide (HMPA) within and in the vicinity of the vermilion locus of Drosophila melanogaster that produce a vermilion mutant phenotype was analyzed after exposure of postmeiotic male germ cells. Mutagenized males were mated to either females wild-type (exr(+)) for nucleotide excision repair (NER) or to females having a deficiency (exr(-)) for NER. Rearrangements, mostly deletions, represented by far the most frequent type of mutational events induced by HMPA that are detected as vermilion mutations. In the exr(+) group, all but one (a double substitution) of 21 mutants characterized were large sequence changes: we found 5 intra-locus deletions, 3 intra-locus deletions associated with insertions and 12 multi-locus deletions. When taken together, deletions and deletion/insertion mutations represent 96% of the HMPA-induced DNA modifications obtained under proficient repair conditions. Of the 10 mutants obtained from crosses with exr(-) females, 6 intra-locus and 2 multi-locus deletions were found, as opposed to just 1 point mutation and 1 double substitution. The ``hypomutability effect' observed with exr(-) genotypes in relation to the wild type seems to be caused by a decrease in the frequency of multi-locus deletions in the former group. The results suggest that the NER system is involved in the generation of multi-locus deletions, whereas intra-locus deletions appear to be formed through a postreplication slipped-misrepair pathway. It is concluded that an eukaryotic in vivo system with no limitations for the recovery of multi-locus deletions, such as vermilion, should be used for the analysis of DNA damage induced by cross-linking agents.