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Karyotype of banded chromosomes of D. discoideum showing chromosomes with (a) separated and (6) unseparated chromatids. For both (a) and (b) two well banded examples of each chromosome are presented; these were obtained from several different cells. In addition, two examples (a) and one example (b) of cells with all chromosomes identifiable are shown. The banding treatments were 0.25 % (w/v) trypsin in 0.85 % (w/v) NaCl for 70 s and 60 s respectively for the two cells in (a), and 0.25 % (w/v) trypsin plus 0.025 % (w/v) EDTA in BSSR for 60 s for the cell in (6). The chromosomes are oriented such that the centromere is uppermost. Bar markers represent 2 pm.
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SUMMARY All seven mitotic chromosomes of the haploid cellular slime mould Dictyostelium discoideum have been identified and numbered using Giemsa banding techniques. Although size differences are not marked, the size distribution is one long, three medium length, and three shorter chromosomes. Each chromosome has a distinctive banding pattern. All...
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... general neither use of Giemsa stain at pH 9, nor incubation in 2 x SSC over a range of temperatures (60 to 75 "C) for a range of times (10 to 90 min) before Giemsa staining, produced consistent banding. In some cases use of BSSR for 2 min at room temp- erature before staining gave successful banding, as did a combination of trypsin, BSSR and EDTA (Fig. 2b). By far the most successful and consistent G-banding technique in- volved trypsin treatment before Giemsa staining. The best conditions were 0.25 % (w/v) trypsin for 60 s at room temperature, followed by the standard staining procedure. Trypsin treatment for less than 60 s resulted in progressively less banding, whereas treatment for ...
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... and incomplete effect. Although really satisfactory preparations have not been achieved, treatment with freshly prepared 5 % (w/v) Ba(OH), for 10 min at 45 to 50 "C followed immediately by incubation in 2 x SSC at 65 "C for 30 min, seems promising. Sometimes treatment with trypsin gave preparations which looked more like C-than G-banding (Fig. ...
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... characteristic banding pattern and manner of chromatid separation were the most useful features for identifying the chromosomes (Fig. 2). Difference in chromosome length was also helpful, but could not always be ,relied on since in late prophase and even early metaphase the differences were small (Fig. 4 b and c). Although early prophase chromosomes were considerably longer and had more bands, the problems of irregular rates of contraction and chromosomal connexions ...
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... late prophase and even early metaphase the differences were small (Fig. 4 b and c). Although early prophase chromosomes were considerably longer and had more bands, the problems of irregular rates of contraction and chromosomal connexions (see below) made it much easier to identify the karyotype using late prophase and early metaphase chromosomes (Fig. 2). A difficulty encountered previously (Brody & Williams, 1974) was that some prophases contained a short chromo- some while others did not. This is most probably explained by differential rates of condensa- tion; chromosome 5, in particular, was observed to condense sooner than the other six chromosomes in several prophases (Fig. ...
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... chromatids of chromosomes I , 2 , 3 , 4 and 5 have been seen to be joined at one end only; sister chromatid separation is less well established in the smaller chromosomes 6 and 7, although they too appear to have terminal centromeres. Representative metaphases showing banded chromosomes with or without separated chromatids, are presented in Fig. 2 . Chromosome I . This is the longest chromosome. It is usually bent close to the centromere and again about two-thirds distal to the centromere, giving it a 'squiggly' appearance. In banded preparations up to five bands uniformly spaced along its length are observed Figs zb and 3). The terminal and subterminal bands at the centromeric ...
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... 2. This is slightly shorter than chromosome I and similar in size to chromo- somes 3 and 4. When chromatids are observed they are commonly crossed distal to the centro- mere (Fig. 2a). Banded preparations usually show three major bands, two of which (situated at or near either end of the chromosome) are prominent, while the third, midway between them, is more lightly stained (Fig. 3). It is important to note that while the band distal to the centromere is distinctly terminal, the band at the centromeric end is ...
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... 3. This is most easily recognized by the chromatids flaring from a point about two-thirds distal to the centromeric end. This partial chromatid separation is often observed before chromatid separation in the other long chromosomes (Fig. 2b). In banded preparations four bands are observed (Fig. 3). The two bands close to the centromere may fuse to form a heavily stained bar. The other two bands are less distinct and are located at the centre and the end of the chromosome distal to the centromere. Chromosome 4. At first glance this chromosome can be confused with chromosome ...
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... a heavily stained bar. The other two bands are less distinct and are located at the centre and the end of the chromosome distal to the centromere. Chromosome 4. At first glance this chromosome can be confused with chromosome 2. Typically the chromatids separate initially at the centre of the chromosome and form a distinctive hollow lens shape (Fig. 2a). Banded preparations show two heavily stained regions at either end of the chromosome (Fig. 3). This clearly distinguishes it from chromo- some 2 which has only one terminal band (Fig. 2 b). There is a third band that is less heavily stained situated close to the centromeric end (Fig. ...
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... can be confused with chromosome 2. Typically the chromatids separate initially at the centre of the chromosome and form a distinctive hollow lens shape (Fig. 2a). Banded preparations show two heavily stained regions at either end of the chromosome (Fig. 3). This clearly distinguishes it from chromo- some 2 which has only one terminal band (Fig. 2 b). There is a third band that is less heavily stained situated close to the centromeric end (Fig. ...
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... 5. This is generally the first to condense and sometimes does so well in advance of the others (Fig. 4a) Chromosome 6. This is the most variable in appearance but is commonly recognizable by its shallow S-shape (Fig. 2). The position of the centromere is not clear, although there is some indication that it is terminal (Fig. 2a). Banded preparations usually show either two or three bands (Fig. 3). , Chromosome 7 . This is the shortest of the chromosomes at metaphase (Fig. 2b) but is quite long before condensation (Fig. 4b and c). In banded ...
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... 5. This is generally the first to condense and sometimes does so well in advance of the others (Fig. 4a) Chromosome 6. This is the most variable in appearance but is commonly recognizable by its shallow S-shape (Fig. 2). The position of the centromere is not clear, although there is some indication that it is terminal (Fig. 2a). Banded preparations usually show either two or three bands (Fig. 3). , Chromosome 7 . This is the shortest of the chromosomes at metaphase (Fig. 2b) but is quite long before condensation (Fig. 4b and c). In banded preparations, when fully con- densed, it is easily recognizable as a darkly stained spot with a pale tail (Fig. 3). ...
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... is the most variable in appearance but is commonly recognizable by its shallow S-shape (Fig. 2). The position of the centromere is not clear, although there is some indication that it is terminal (Fig. 2a). Banded preparations usually show either two or three bands (Fig. 3). , Chromosome 7 . This is the shortest of the chromosomes at metaphase (Fig. 2b) but is quite long before condensation (Fig. 4b and c). In banded preparations, when fully con- densed, it is easily recognizable as a darkly stained spot with a pale tail (Fig. 3). Chromatid separation is observed but the small size makes positioning of the centromere difficult, although it is most probably terminal (Fig. ...
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... at metaphase (Fig. 2b) but is quite long before condensation (Fig. 4b and c). In banded preparations, when fully con- densed, it is easily recognizable as a darkly stained spot with a pale tail (Fig. 3). Chromatid separation is observed but the small size makes positioning of the centromere difficult, although it is most probably terminal (Fig. ...
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... what we believe to be sister chromatids, partially separated both in late prophase and early metaphase. These reports were published when little was known of the nuclear behaviour of D. discoideum, and the photographs were interpreted as showing paired homo- logues at meiosis. Although the chromosomes showing chromatid twisting and separation (Fig. 2 a) superficially resemble meiotic preparations of higher organisms, such a resemblance I99 is clearly misleading. Only haploid amoebae dividing by binary fission were used in this study. There is genetic evidence that in two closely related species of slime moulds (Dictyo- stelium mucoroides and Dictyostelium giganteum) meiosis occurs in ...
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... level of nuclear organization, such as has been suggested in yeast (Lauer & Klotz, 1975;Wintersberger et al., 1975)~ remains to be established. It seems that if there are connexions, they break up after the chromosomes become arranged on the metaphase plate. In most ,cases seven individually recognizable chromosomes are observed at metaphase (Fig. 2). When fewer than seven chromosomes are observed, invariably this is caused by ambiguous overlap of chromosomes, or association between two chromosomes which can be recognized as individuals using banding techniques (Fig. 4c, d, e). Hence we conclude that D. discoideum has seven chromosomes. We believe that the recent suggestion by ...
Citations
... Other linkage groups were rapidly defined by other markers until a total of 6 linkage groups were well established (Newell et al. 1993). Each linkage group appeared to define a chromosome since chromosomal stains of haploid strains indicated the presence of 7 chromosomes (more about the seventh "chromosome" later) (Sussman, 1961;Robson and Williams, 1977;Sucgang et al., 2003). At this point renewed efforts were made to better understand the sexual cycle so that the high frequency of recombination that is usually associated with meiosis could be harnessed to order the markers along the chromosomes. ...
The 34 Mb genome of Dictyostelium discoideum is carried on 6 chromosomes and has been fully sequenced by an international consortium. The sequence was assembled on the classical and physical maps that had been built up over the years and refined by HAPPY mapping. Annotation of the sequence predicted about 12,000 genes for proteins of at least 50 amino acids in length. The total number of amino acids encoded (the proteome) is more than double that in yeast and rivals that of metazoans. The genome sequence shows all the proteins available to Dictyostelium as well as definitively showing which domains have been lost since Dictyostelium diverged from the line leading to metazoans. Genomics opens the door to determining the expression patterns of all the genes during growth and development using microarrays. This approach has already uncovered a wealth of new markers for the stages of development and the various cell types. Transcription factors and their cis-regulatory sites that account for the surprising complexity of Dictyostelium development can be analyzed much more easily now that we have the complete sequence.
... In each case, seven mitotic chromosomes were clearly visible. Although older data had concluded that Dictyostelium contains seven chromosomes [2,[21][22][23], it has been difficult to find markers in more than six linkage groups, leading to a consensus that only six bona fide chromosomes exist [24][25][26]. This argument has recently been addressed by Sucgang et al. (2003), who conclude that one of the seven chromosomes seen by DAPI staining derives from multiple copies of the ribosomal DNA aggregated into a single, chromosome-sized body which disintegrates during FISH [27]. ...
Normally, vegetative Dictyostelium grow as haploid cells. Occasionally, two haploid cells fuse together during normal growth, forming a diploid cell containing both parental sets of chromosomes within a single nucleus. The diploid state is reasonably stable, and the growth, development, and general behavior of diploids are similar to their haploid parents. However, during normal growth of diploids, cells may spontaneously lose one copy of each chromosome at random and revert back to a haploid state containing a selection of chromosomes from both parents. This diploid cycle therefore allows nonsexual recombination between two different mutant strains.
Diploid cells have multiple practical uses. They allow the generation of double and multiple knockouts, and are particularly useful for strains that are sick or difficult to generate using molecular genetics. They provide a means of manipulating genes that are lethal when disrupted in haploids. In diploids, it is possible to isolate heterozygous knockouts with no phenotype and then introduce a further mutant allele. These cells can then be segregated to yield haploid progeny with an effective gene replacement. Similarly, diploids made from different parent strains offer a means of examining the effects of different genetic backgrounds and overriding strain-specific phenotypes. A number of other uses are possible, making parasexual genetics potentially even more versatile.
... Normal haploid Dictyostelium discoideum amoebae contain seven chromosomes, as revealed by cytogenetical studies [1][2][3][4]. However, available genetic techniques based on the parasexual cycle in Dictyostelium have allowed assignment of developmental, behavioural and other loci to only six of the expected seven linkage groups in this organism [5][6][7]. ...
During the course of mapping and complementation analysis of phototaxis (pho) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK. These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.
... Laboratory strains ofD. discoideum are thought to have seven chromosomes (6,7), although only six linkage groups have been identified (3). ...
... The genetic literature suggests that there are six linkage groups in commonly used D. discoideum strains (3), although cytological examination of metaphase preparations reveals seven (6,7). Our results clearly show six, although it is difficult to rule out that there is a seventh, even with the use of randomly chosen cDNA probes, since there could be a seventh transcriptionally silent chromosome. ...
This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.
... Classical genetic analysis of D. discoideum includes cytological studies of its 7 chromosomes in haploid strains (21), as well as the preparation of a genetic map, containing over 100 genes mapped to a particular chromosome (22). This organism has a total genome size of 55 megabases (23), about 8 megabases of which is in the form of -90 copies of a 88 kilobase, linear ribosomal DNA molecule (24). ...
Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields)) into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1–10 Mb), and −90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (−2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA.
This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D.discoideum . Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D.discoideum plasmid Ddpl had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D . discoideum genome.
... have determined that most spindle MTs are nkMTs. Taking account of the seven chromosomes of D. discoideum (e.g., see Robson & Williams, 1977) and two to three kMTs per chromosome (Moens, 1976; and our own unpublished observations), the predicted 14-21 kMTs are only a fraction of the 140-160 MTs seen in these halfspindles. The nkMTs are of diverse lengths: many are short and end between pole and equator, whereas a few extend all the way from one pole to the immediate vicinity of the other. ...
Ten mitotic spindles from Dictyostelium discoideum have been studied by electron microscopy of serial sections. We have used computer graphics to track individual microtubules (MTs) in three dimensions and to compare seven spindles at different stages of anaphase and telophase. The central spindle of early anaphase is formed by the interdigitation of two sets of pole-associated MTs. The distribution of MT lengths at this stage is hetero-disperse. During anaphase total MT length decreases by a factor of about 2 as a result of two opposing changes in MT length: the longer MTs that interdigitate become even longer, while the short MTs, including those attached to kinetochores, become shorter still and decrease in number. The extent of MT interdigitation is less in longer spindles than in short ones. In metaphase and early anaphase, the MTs are not in an ordered arrangement as seen in spindle cross-sections, but as anaphase proceeds the MTs cluster into a square-packed, paracrystalline bundle in which most of the nearest neighbours come from opposite poles. This arrangement and the condensation-like increase in order suggest the existence of specific interactions between antiparallel MTs. A quantitative analysis of MT positions supports this interpretation, but direct evidence for convincing bridges between MTs is lacking. The pole-distal ends of the MTs that interdigitate show an irregular termination (C-shaped ends in transverse view), as is characteristic of MTs that are either adding or losing subunits. Since it is these interdigitating MTs that elongate, and since the shortening MTs show the customary blunt endings, we conclude that subunits add to the interdigitating MTs at their pole-distal ends. This inference, combined with other structural data, suggests that the interdigitating MTs of Dictyostelium are sliding over one another as they polymerize in anaphase. It also suggests a simple model for why the spindle becomes thinner as it elongates. We propose that MT interdigitation defines a region where MTs bind a factor that will associate only with antiparallel MTs. This factor biases the MT assembly equilibrium toward polymer. As the shorter MTs slide out of this region, they lose their polymerization advantage and depolymerize, releasing subunits to contribute to the further elongation of the already longer MTs. The properties of the Dictyostelium spindle are compared with those of both higher and lower eukaryotes.
... Linkage assignment allows one to divide the genome of D. discoideum into seven regions (since there are seven chromosomes; ROBSON and WILLIAMS 1977). For preliminary genetic analysis this is often adequate. ...
... The centromere is shown on the left as all seven D. discoideum chromosomes appear to be acrocentric ( ROBSON and WILLIAMS 1977). This is consistent with the genetic data although not rigorously proven for loci adjacent to the centromere because of the possibility of mitotic nondisjunction. ...
... In the map presented here, all markers located on a particular linkage group can be placed on a single chromosome arm. This finding is consistent with cytological studies in which all seven D. discoideum chromosomes appeared to be acrocentric (ROBSON and WILLIAMS 1977). Cytological techniques have been used in the establishment of several kinds of chromosomal abnormality (WILLIAMS, ROBSON and WELKER 1980;WELKER and WILLIAMS 1981) and it can be expected that by using a combination of known gene orders and cytological study many new chromosomal rearrangements will soon be found. ...
A genetic map of the cellular slime mold Dictyostelium discoideum is presented in which 42 loci are ordered on five of the seven linkage groups. Although most of the loci were ordered using standing mitotic crossing-over techniques in which recessive selective markers were employed, use was also made of unselected recombined haploid strains. Consistent with cytological studies in which the chromosomes appear to be acrocentric, only a single arm has been found for each of the five linkage groups studied. The mating-type locus, matA, has been located in the tsgE-sprA interval on linkage group I on the basis of studies on diploids formed between strains of opposite mating type that have escaped from vegetative incompatibility.
... In general, interphase nuclei of P. pallidum were more intensely stained than those of similar preparations of D. discoideum, so that trypsin treatment to prevent cytoplasmic staining (Robson & Williams, 1977) was usually unnecessary. PoZysphondylium pallidurn ANU122 showing 11 and 12 chromosomes, respectively. ...
... The axenically grown P. pallidum amoebae examined here contained multinucleate cells, thus exhibiting similar behaviour to D. discoideum amoebae grown in this medium (Robson & Williams, 1977;Zada-Hames & Ashworth, 1978 ;Sameshima et al., 1978). Polysphondyliurnpallidum strain WS320 had multinucleate cells when grown in a different axenic medium (Hohl & Raper, 1963), suggesting that multinucleate cells may be a feature of axenic cultures of cellular slime moulds. ...
... It is also important to use short incubation times with the benzimidazole derivatives, as large numbers of aneuploid and diploid metaphases are produced on prolonged incubation (Welker & Williams, 1980). In order to determine the chromosome number of P. pallidum with certainty, both karyotype analysis (Robson & Williams, 1977) of those P. pallidum isolates examined so far, and further studies on different isolates are needed. There are clearly more chromosomes than in D. discoideum (n = 7 ) and the number is likely to be n = 11 or 12; possibly the 11 chromosome isolates have two chromosomes fused. ...
Techniques used previously to examine the mitotic chromosomes of Dictyostelium discoideum have been applied to the cellular slime mould Polysphondylium pallidum. Mitosis in P. paElidurn is similar to that in D. discoideum except that a metaphase plate configuration is rare and that it has more chromosomes (n = 11 or 12) than D. discoideum (n = 7). Some metaphase arrest (mitotic index 14 yo) was achieved in P. pallidum when colchicine was used at a high concentration (10 mg ml-l). Low concentrations of benzimidazole derivatives jthiabendazole, cambendazole, ben late and nocodazole) caused the arrest of cell division, mitotic indices greater than 50 %, chromosome doubling and lowered plating efficiency. Growth of P. pallidum in axenic medium was investigated.
... Several studies have recently been aimed at determination of the karyotype of the cellular slime mould Dictyosteliurn discoideum (Robson & Williams, 1977 ;Zada-Hames, 1977 ;Brody & Williams, 1974;Sussman, 1961). These investigations have been hampered, however, by the inability to obtain large numbers of cells in metaphase. ...
... These investigations have been hampered, however, by the inability to obtain large numbers of cells in metaphase. While the number of cells in metaphase can be increased by treatment with colchicine at high concentrations (Zada-Hames, 1977) or by release from starvation (Robson & Williams, 1977), these techniques have not greatly facilitated routine chromosomal studies particularly with cells growing on bacteria. ...
... Amoebae were fixed in methanol/acetic acid (3: 1, v/v) and air-dried on to slides as described previously (Brody & Williams, 1974). easily observed by light microscopy (Robson & Williams, 1977). The mitotic index was determined by examining all nuclei in a series of randomly chosen fields; at least 1000 nuclei were examined for each sample. ...
Thiabendazole and cambendazole induced mitotic arrest and isogenic diploid formation through chromosome doubling in amoebae of the cellular slime mould Dictyostelium discoideum grown either axenically or in a bacterial suspension. The effect of nocodazole on axenically grown cells was similar to that of thiabendazole and cambendazole, but with cells grown on bacteria nocodazole was a much less effective inducer of mitotic arrest and did not lead to isogenic diploid formation. Benomyl, the active ingredient of ben late, had little or no effect on the induction of mitotic arrest and was a poor inducer of isogenic diploids in cells grown either axenically or on bacteria. All four benzimidazole derivatives were effective in haploidizing pre-existing diploids.
... discoideum is one of the lowest of any eukaryotic organism [1], chromosomes are small and not easily studied [2], and metaphase blocking of cell division is difficult to obtain, due to the high resistance to antimitotic drugs shown by this organism [3,4]. Despite these limiting factors, chromosomal studies have been recently conducted using high concentrations of the mitotic inhibitor colchicine [4], or the release from starvation conditions [5] to increase the mitotic index. ...
... The metaphasearrested cells which can be reproducibly obtained with 5-7/ag/ml for 7-8 h incubation with nocodazole are about 50%. This figure is much higher than either the 11% reached with colchicine treatment for 14-16 h [4] or the 6% than can be seen by releasing amoebae from starvation on filters [5]. ...
