Further Evidence for Lack of Gene Expression in the Tetrahymena Micronucleus

Certain galA mutations in the ciliated protozoan Tetrahymena thermophila confer an almost total loss of galactokinase activity in homozygotes. Heterokaryons have been constructed that are homogeneous for the galA1 mutation in the (45n) macronucleus, but which contain a galA⁺ (2n) micronucleus. Soluble cell extracts prepared from these heterokaryons have been assayed for galactokinase activity, using a radiometric assay for the conversion of galactose to galactose-1-phosphate (gal-1-P). No galactokinase activity attributable to the micronuclear genes is observed in such heterokaryons. These results, obtained with the galA1 marker, provide the first direct, quantitative evidence for the lack of micronuclear (germ line) gene expression in Tetrahymena during vegetative growth, and substantiate the predictions of previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis. The generality of this conclusion will be established in the future when other enzymically assayable mutations become available for similar studies.     

驱动蛋白Kin11调控嗜热四膜虫生殖系小核的减数分裂

驱动蛋白(kinesin)是分子马达蛋白质超家族成员,主要参与囊泡与细胞器的运输、纺锤体组装、有丝分裂和减数分裂等过程。在减数分裂期,不同驱动蛋白发挥功能的调控机制并不十分清楚。嗜热四膜虫(Tetrahymena thermophila)中含有14个驱动蛋白家族成员。其中,kinesin-6家族的唯一成员Kin11(TTHERM_00637750),在营养生长期低表达,饥饿期不表达,有性生殖期表达上调。Kin11编码1608个氨基酸,包含1个N端保守的马达蛋白结构域,C端卷曲螺旋(coiled-coil)结构域,并在N端和C端分别含有核定位信号NLS1和NLS2。Kin11在营养生长期和有性生殖期,定位在有丝分裂和减数分裂的小核和纺锤体上,并在有性生殖后期alignment阶段定位于小核上。Kin11与微管蛋白共定位于有丝分裂和减数分裂的纺锤体上。将Kin11的N端含有NLS1的1~400位氨基酸序列截短后,截断突变体定位在有性生殖减数分裂期的小核和纺锤体上。而将其C端含有NLS2的1008~1608位氨基酸残基截短后,截断突变体只能定位在有丝分裂和减数分裂后期的小核及有丝分裂的纺锤体上。敲除KIN11导致减数分裂过程中的纺锤体结构发生异常变化,小核染色体不均等分离与丢失,有性生殖发育停滞。结果表明,嗜热四膜虫驱动蛋白Kin11通过影响纺锤体结构,参与调控四膜虫生殖系小核在减数分裂过程中的正常分离。     

Alternative Processing of Sequences During Macronuclear Development in Tetrahymena thermophila1

ABSTRACT. DNA is eliminated during development of the somatic MACronucleus from the germinal MICronucleus in the ciliated protozoan, Tetrahymena thermophila. Facultatively persistent sequences are a class of sequences that persist in the MAC DNA of some cell lines but are eliminated from the MAC DNA of other cell lines. One cloned MAC fragment contains a persistent sequence as well as sequences normally retained in the MAC. When this cloned fragment was used to construct MAC restriction maps of this region in cell lines whose MAC DNAs do, or do not, contain the persistent sequence, extensive variation in the map flanking this region was observed. The different DNA rearrangements of this MIC segment are epigenetically determined during or soon after MAC development. Moreover, different rearrangements may occur among the 45 copies of this MIC segment as a MAC is formed, resulting in polymorphisms that are later resolved by phenotypic assortment.     

Flanking Regulatory Sequences of the Tetrahymena R Deletion Element Determine the Boundaries of DNA Rearrangement

In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement.     

上海四膜虫和两株嗜热四膜虫的rRNA基因ITS-1序列及分子系统关系

测定并比较了自接型的上海四膜虫（Tetrahymena shanghaienisis)和两株接合型的嗜热四膜虫（T.thermopddhilaⅡ和T.thermophilaⅥ）的ITS-1序列,以多态嗽叭虫（Stentor polymorphrus)为外来群,利用最大简约法和邻接法构建了它们的系统发育树。分析指出：三者中,T.shanghaienisis较早地从祖先种中分化出来;自接型可能是一种较接合型原始的生殖方式。     

A Genetic Screen for Vegetative Gene Expression in the Micronucleus of Tetrahymena thermophila

ABSTRACT. The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila . A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization. Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization. This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle.     

Cytologic and Autoradiographic Studies of the Micronucleus at Meiotic Prophase in Tetrahymena pyriformis

Micronuclear changes of variety 1 of Tetrahymena pyriformis during meiotic prophase have been observed by the light microscope. Morphologic changes in the micronucleus are divided into 6 stages. In stage I, chromatin begins to polarize; in stage II, the micronucleus becomes spindle shaped; and in stage III, one end of the micronucleus protrudes to form a “neck.” In stage IV, where the micronucleus elongates to maximal length, the whole micronucleus consists of 2 chromatin threads pairing longitudinally. One thread probably contains one genome. In stage V, the elongated thread becomes shorter and thicker. Finally, in stage VI, separate chromosomes appear and enter into metaphase. To discover the role of the elongation of the micronucleus, called crescent formation, autoradiographic analysis of RNA and DNA synthesis were undertaken using [³H]uridine and [³H]thymidine. Pulse label and chase experiments show that the crescent in stages II and III is actively synthesizing RNA. Though no remarkable DNA synthesis was observed during meiosis, a small amount of DNA synthesis occurred during the 1st and 2nd prezygotic divisions.     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

Localization of 3H-histamine in the nucleus of Tetrahymena

3H-histamine was incorporated by Tetrahymena partly into membrane-associated structures (cilia), partly into intra-cytoplasmic localizations, and above all into food vacuoles. A considerable amount appeared in the nucleus, in practically exclusive association with the heterochromatin.     

Genomic Organization and Developmental Fate of Adjacent Repeated Sequences in a Foldback DNA Clone of Tetrahymena thermophila

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

Chromosomal Localization of an Exocytosis Mutant in Tetrahymena thermophila

ABSTRACT. Exocytosis mutants of Tetrahymena thermophila are deficient in mucus release. Experiments to chromosomally locate two of these mutants are described, using the technique of deletion mapping with nullisomic strains. One exo locus has been assigned to chromosome 5.     

Localization of DNA Sequences Tightly Associated with the Synaptonemal Complex in Compositional Fractions of the Golden Hamster Genome*

Synaptonemal complex (SC) isolated from spermatocyte nuclei after their exhaustive hydrolysis by DNase II contains DNA sequences tightly associated with it (SCAR DNA). Here, the compositional properties of a cloned family of golden hamster SCAR DNA were studied. For this purpose, 27 SCAR DNA clones were hybridized with compositionally fractionated golden hamster genomic DNA. The sequences of the SCAR DNA family were mainly localized in the GC-poor isochore families L1 and L2, which accounted for 63% of hybridization signals. The remaining 37% of signals pertained to the GC-rich isochore families H1 and H2. Thus, SCAR DNA proved to be distributed throughout the genome, irrespective of differences in density and sequence type between isochore families. Moreover, the SCAR DNA sequences containing the regions of homology with LINE/SINE repeats were found in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that the SC and SCAR DNA participate in chromatin reorganization during meiosis prophase I, which should result in the attachment of chromatin loops to the lateral elements of SC throughout its length.