Kinetochore fiber formation in animal somatic cells: dueling mechanisms come to a draw

The attachment to and movement of a chromosome on the mitotic spindle are mediated by the formation of a bundle of microtubules (MTs) that tethers the kinetochore on the chromosome to a spindle pole. The origin of these “kinetochore fibers” (K fibers) has been investigated for over 125 years. As noted in 1944 by Schrader [Mitosis, Columbia University Press, New York, 110 pp.], there are three possible ways to form a K fiber: (a) it grows from the pole until it contacts the kinetochore, (b) it grows directly from the kinetochore, or (c) it forms as a result of an interaction between the pole and the chromosome. Since Schrader's time, it has been firmly established that K fibers in centrosome-containing animal somatic cells form as kinetochores capture MTs growing from the spindle pole (route a). It is now similarly clear that in cells lacking centrosomes, including higher plants and many animal oocytes, K fibers “self-assemble” from MTs generated by the chromosomes (route b). Can animal somatic cells form K fibers in the absence of centrosomes by the “self-assembly” pathway? In 2000, the answer to this question was shown to be a resounding “yes.” With this result, the next question became whether the presence of a centrosome normally suppresses K fiber self-assembly or if this route works concurrently with centrosome-mediated K-fiber formation. This question, too, has recently been answered: observations on untreated live animal cells expressing green fluorescent protein-tagged tubulin clearly show that kinetochores can nucleate the formation of their associated MTs in a unique manner in the presence of functional centrosomes. The concurrent operation of these two “dueling” routes for forming K fibers in animal cells helps explain why the attachment of kinetochores and the maturation of K fibers occur as quickly as they do on all chromosomes within a cell.     

Karyotypes and geographical distribution of ricefishes from Yunnan,southwestern China

Morphometric and karyotypic studies were made on two species of ricefishes collected from Yunnan, southwestern China.Oryzias latipes from Yunnan had the same morphological and karyological characteristics asO. latipes collected from eastern China. The Yunnan populations had 2n, 46 chromosomes consisting of 3 metacentric, 8 submetacentric, 2 subtelocentric, and 10 acrocentric pairs, the arm number (NF) being 68 (2n=46, NF=68, 3M+8SM+2ST+10A). The karyotype was characterized by having a “large” metacentric pair and nucleolus organizer regions (NORs) on the short arms of a submetacentric pair.Oryzias minutilius from Yunnan had the same morphological characteristics asO. minuiillus from Thailand and Burma, although the karyotype was different from that collected from Thailand. The Yunnan population had 2n, 42 chromosomes consisting of 21 acrocentric pairs, NF being 42 (2n=42, NF=42, 21A). The karyotype was characterized by having NORs at the telomeric regions of an acrocentric pair.Oryzias latipes occurs widely on the eastern Yunnan Plateau where the climate is temperate or subtropical, whereasO. minutilius is found in Xishiangbanna, the southern low mountain areas of Yunnan, where the climate is tropical.     

Karyotypes of two Antarctic fishes,Notothenia gibberifrons andNotothenia coriiceps neglecta

The chromosomes of two species of Antarctic fishes,Notothenia (Gobionotothen) gibberifrons andNotothenia (Notothenia) coriiceps neglecta, were prepared by the air-drying method at the Polish Antarctic Station “Henryk Arctowski” during the austral summer 1984–1985. ForN. (G.) gibberifrons the diploid number is2n = 46 consisting of 2 metacentric (m) pairs, 1 submetacentric (sm) pair and 20 telocentric (t) or subtelocentric (st) pairs. ForN. (N.) coriiceps neglecta the diploid number is 2n = 22 consisting of 9 m pairs, 1 sm pair and 1st pair. Some aspects of karyological evolution of these fishes are discussed.     

Noncanonical meiosis in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as a model for studying the molecular bases of the homologous chromosome synapsis,crossing over,and segregation

The nematode C. elegans is a classic study object of developmental biology and genetics, which is particularly suitable for studying the molecular bases of meiosis. Developing meiocytes are located in the threadlike gonads of C. elegans in linear gradient order of the stages of meiosis, which facilitates studying the order of intracellular events during meiosis. C. elegans has polycentric chromosomes. This causes a special order of events during meiosis, and as a consequence, meiosis in C. elegance differs from canonical meiosis of most eukaryotes. In the meiotic prophase I, all chromosomes carry single protein “pairing centers.” They are responsible for joining homologous chromosomes in pairs. This initiates the formation of synaptonemal complexes (SCs). Programmed double-stranded DNA breaks appear after initiation of the SC assembly, and they give rise to meiotic recombination. The initiation of meiotic recombination after the chromosome pairing distinguishes the C. elegans meiotic pattern from those in the absolute majority of eukaryotes studied. C. elegans has strict crossing over interference, which allows for the formation of one chiasma per bivalent. In the late prophase I, the polycentric centromeres are remodeled, one of the chromosome ends acquires a cuplike kinetochore, and during two meiotic divisions, chromosomes behave as monocentric. The study of meiosis in C. elegans allows for separate investigation of synapsis and recombination of homologous chromosomes and provides material for studying the evolution of meiosis.     

Cytogenetics of the parthenogenetic grasshopper Warramaba virgo and its bisexual relatives

Combinations of DNA-binding fluorescent dyes and counterstains that enhance selectivity and contrast in primary stain fluorescence were used to differentiate types of C-bands in the genus Warramaba. Chromomycin A₃ (in conjunction with two A-T binding counterstains), which identifies chromosome segments enriched in G-C base pair clusters, stains only a minority of the C-bands in Warramaba species, but these include all those known to contain 18S + 26S rRNA cistrons and most of those containing 5S rRNA genes. DAPI/actinomycin D fluorescent staining is positive for a very few bands, including two (in the Standard phylad of W. virgo) that are at or adjacent to sites containing 5S rRNA cistrons. One of the latter regions is also positively stained by DAPI/distamycin A which, in addition, highlights some centromeric bands. The fluorescent staining patterns of the Standard and Boulder-Zanthus phylads of W. virgo are significantly different, confirming their independent origin by hybridization between different races of the ancestral species “P169” and “P196”.     

Microsatellite loci variation and investigation of gene flow between two karyoforms of <Emphasis Type="Italic">Cricetulus barabensis</Emphasis> sensu lato (Rodentia,Cricetidae)

We examine the diversity of six microsatellite loci and partial RAG1 exon of “barabensis” and “pseudogriseus” karyoforms in Cricetulus barabensis sensu lato species complex. A total of 435 specimens from 68 localities ranging from Altai to the Far East are investigated. The results of the population structure analysis (factor analysis and NJ tree based on Nei genetic distances) support subdivision into two well-differentiated clusters corresponding to the two karyoforms. These karyoforms are also well differentiated by the level of microsatellite variability. In several “barabensis” specimens, we found microsatellite alleles that are common in “pseudogriseus” populations but are otherwise absent in “barabensis.” Most of these specimens originate from a single population in one of the zones of potential contact between karyoforms, Kharkhorin in Central Mongolia. These molecular results are consistent with previously published karyological data in suggesting that rare hybridization events between the two chromosomal races occur in nature.     

A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages

Background

The C3bot1 protein (∼23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.

Methodology/Principal Findings

Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.

Conclusions/Significance

In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.     

Mechanism of haploidy-dependent unreductional meiotic cell division in polyploid wheat

Unreductional meiotic cell division (UMCD) generates unreduced gametes and leads to polyploidy. The tetraploid wheat “Langdon” (LDN) undergoes normal meiosis, but its polyhaploid undergoes UMCD. Here, we found that sister kinetochores oriented syntelically at meiosis I in LDN, but amphitelically in LDN polyhaploid and the interspecific hybrid of LDN with Aegilops tauschii. We also observed that sister centromere cohesion persisted until anaphase II in LDN, LDN polyhaploid, and the interspecific hybrid. Meiocytes with all chromosomes oriented amphitelically underwent UMCD in LDN polyhaploid, and the interspecific hybrid, suggesting the tension created by the amphitelic orientation of sister kinetochores and persistence of centromeric cohesion between sister chromatids at meiosis I contribute to the onset of UMCD. Most likely, some ploidy-regulated genes were responsible for kinetochore orientation at meiosis I in LDN and LDN-derived polyhaploids. In addition, we found sister kinetochores of synapsed chromosomes oriented syntelically, whereas asynapsed chromosomes oriented either amphitelically or syntelically. Thus, synapsis probably is another factor for the coordination of kinetochore orientation in LDN.     

“Uno,nessuno e centomila”: the different faces of the budding yeast kinetochore

“One, no one and one hundred thousand” is a masterpiece of Italian literature, written by Luigi Pirandello. The central theme is that in each individual there are multiple personalities, since one’s perception of one’s self differs from the view of others. As a consequence, a unique identity does not exist, but rather one hundred thousand. This concept can be very well applied to the kinetochore, one of the largest macromolecular complexes conserved in eukaryotes. The kinetochore is essential during cell division and fulfills different sophisticated functions, including linking chromosomes to spindle microtubules and delaying anaphase onset in case of incorrect bi-orientation. In order to perform these tasks, the kinetochore shapes its structure by recruiting different subunits, such as the components of the spindle assembly checkpoint (SAC) or the monopolin complex during meiosis. It also modifies its internal organization by rearranging intramolecular connections and acquiring a distinct identity at different time points of cell division. In this review, we describe recent insights into the changes in composition and configuration of the kinetochore in mitosis and meiosis, focusing on the kinetochore of Saccharomyces cerevisiae.     

Identification of the centromeres of Leishmania major: revealing the hidden pieces

Leishmania affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in Trypanosoma brucei (KKTs) leads to the identification of a Leishmania KKT gene candidate (LmKKT1). The GFP‐tagged LmKKT1 displays “kinetochore‐like” dynamics of intranuclear localization throughout the cell cycle. By ChIP‐Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of Leishmania chromosomes opens avenues to explore their role in mosaic aneuploidy.     

Karyosystematic studies on threeCrepis species (Asteraceae) endemic to Greece

This work examines the cytogeographical distribution, the morphological characters, and the karyotypes of threeCrepis species endemic to Greece (C. sibthorpiana, C. incana, andC. heldreichiana). C. sibthorpiana is diploid (2n = 2x = 8),C. incana is diploid (2n = 2x = 8) and tetraploid (2n = 4x = 16, 17), andC. heldreichiana is always dekaploid (2n = 10x = 40). The Giemsa positive bands, usually pairs of dots, are mainly centromeric inC. incana, while they are terminal inC. sibthorpiana (on the short arm of all chromosomes) and inC. heldreichiana (on both arms of all chromosomes). Intercalary C-bands are scarce and usually variable within karyotypes, individuals, and species. The most variable karyotype both in Feulgen and Giemsa preparations is that ofC. incana, in which also supernumerary chromosomes were observed, which are polysomic to standard set members. On the basis of morphological and karyological data the evolutionary relationships among the threeCrepis taxa are discussed.     

DNA replication,G- and C-bands and meiotic behaviour of supernumerary chromosomes of Rattus rattus (Linn.)

Supernumerary chromosomes have been observed in a few individuals of three subspecies of Rattus rattus from India and Nepal. The supernumerary chromosomes are late replicating and positively heteropycnotic during meiosis which characterize their heterochromatic nature. Their G-banding patterns do not exactly resemble the patterns exhibited by the chromosomes of similar size and morphology of the normal complement. The supernumerary chromosomes become conspicuous for the lack of a centromeric C-band in them as compared to the prominent C-bands in other chromosomes of the complement.     

Small chromosomes among Danish Candida glabrata isolates originated through different mechanisms

We analyzed 192 strains of the pathogenic yeast Candida glabrata from patients, mainly suffering from systemic infection, at Danish hospitals during 1985–1999. Our analysis showed that these strains were closely related but exhibited large karyotype polymorphism. Nine strains contained small chromosomes, which were smaller than 0.5 Mb. Regarding the year, patient and hospital, these C. glabrata strains had independent origin and the analyzed small chromosomes were structurally not related to each other (i.e. they contained different sets of genes). We suggest that at least two mechanisms could participate in their origin: (i) through a segmental duplication which covered the centromeric region, or (ii) by a translocation event moving a larger chromosome arm to another chromosome that leaves the centromere part with the shorter arm. The first type of small chromosomes carrying duplicated genes exhibited mitotic instability, while the second type, which contained the corresponding genes in only one copy in the genome, was mitotically stable. Apparently, in patients C. glabrata chromosomes are frequently reshuffled resulting in new genetic configurations, including appearance of small chromosomes, and some of these resulting “mutant” strains can have increased fitness in a certain patient “environment”.     

甘肃紫斑牡丹品种与中原牡丹品种银带和Giemsa C带的研究

分别对甘肃紫斑牡丹品种和中原牡丹品种进行了核型,Ａｇｇｋｐｈｔ Ｇｉｅｎｓａ Ｃ带的研究。发现紫斑牡丹品种核型组成为２ ＝１０＝８ｍ＋２ｓｔ;中原牡丹品种核型组成为２ｎ＝１０＝６ｍ＋２ｓｍ＋２ｓｔ。ＧｉｅｍｓａＣ带带型显示,供试品种均能显示染色体端带,但天染色体端带的数目及分布位置上具品种特异性。     

Somatic deletion and redistribution of telomeric heterochromatin in the genus Secale and in Triticale

C-banding analysis of populations of Secale kuprijanovii L., S. cereale L., and x Triticosecale Wittmack established that Secale chromosomes that were modified by the loss of a telomeric C-band arose spontaneously by breakage in somatic tissue and could be stabilized and maintained over at least two generations. In S. cereale approximately double-sized C-bands were seen on every arm that originally contained a C-band except 1RS, 2RS, 3RS, and 7RS. One plant was homomorphic for an amplified band on 3RL which was stable over two generations. In the tetraploid triticale analyzed, an amplified telomeric C-band was found on 5RS and was stable in the homomorphic condition for two generations. Even though Secale chromosomes with deleted or amplified telomeric C-bands can arise spontaneously in the somatic and germ tissue of Secale species and triticale, they are not common. The conditions required for their formation and stabilization within a population are not known.     

Restrictive localization of centromeric heterochromatin (C-bands) in Presbytis e. entellus (Dufresne) as compared to Macaca mulatta (Zimmerman)

C-bands are observed in the centromeric regions of only three pairs of autosomes and the distal portion of the small acrocentric Y in a total complement of 44 chromosomes of a male Presbytis e. entellus. Simultaneously treated slides of a Rhesus monkey, however, have C-bands in all the 42 chromosomes. The lack of C-bands may be due to (1) absence of highly repetitive DNA in the centromeric region of certain chromosomes or (2) presence of minute quantity of such DNA which is imperceptible or (3) different types of centromeric heterochromatin with a varying degree of repetition of DNA sequences all of which do not react in similar manner to various techniques employed at present. It is hypothesized that the centromeric heterochromatin rich in satellite DNA helps in withstanding the force of excessive coiling of chromosomes at the centromere to facilitate the functioning of the genes for microtubular protein during cell division when other genes are rendered inactive due to compactness of chromosomes.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

B-Chromosomen bei der GattungLeucanthemum (Asteraceae) in der Tschechoslowakei

The taxonomic arrangement and the karyological analysis of 166 populations ofLeucanthemum demonstrates the existence of 5 taxa for Czechoslovakia; they are grouped provisionally intoL. rotundifolium (W. K.) DC. (2n = 18) and the polyploid complexL. vulgare Lam. with “subsp.vulgare” (2n = 18), “subsp.alpicola” (2n = 18), “subsp.ircutianum” (2n = ± 36) and “subsp.pannonicum” (2n = ± 54). B-chromosomes have not been traced in diploids but have turned up in great numbers in tetraploid (21%) and hexaploid populations (20%). Their presence is not apparent from external characters. Diploids and polyploids evidently are selfsterile.     

Heterogeneity of heterochromatin in plants: Comparison of Hy- and C-bands inVicia faba

The comparison of Hy- and C-bands reveals three types of heterochromatin (Hy+, Hy−, and Hy 0) inVicia faba. By C-banding, the total of constitutive heterochromatin is identified. The Hy-banded heterochromatin is restricted to the M-chromosome: The nucleolus-associated heterochromatin appears as two darkly stained (Hy+)-bands bordering the secondary constriction, while reduced staining (Hy−)-bands are located near the centromere. The heterochromatin of the S-chromosomes is of the (Hy 0)-type, i.e., it does not respond to the Hy-banding procedure. A complete karyogram of the C-banded chromosome complement is presented and discussed. A comparison of C-bands and cold-sensitive segments clearly shows that negatively as well as positively reacting cold-induced segments are partly heterochromatic, partly euchromatic.