Journal of Chromosomes

Journal of Chromosomes

Journal of Chromosomes

Current Issue Volume No: 1 Issue No: 1

Research Article Open Access Available online freely Peer Reviewed Citation

The Chromosomes of Dictyostelium Giganteum

Rishikesh Kumar 1 2,   Pooja S. Kulshreshtha 1,   Vidyanand Nanjundiah 1,   Jayarama S. Kadandale 1  

1Centre for Human Genetics, Electronic City (Phase I), Bengaluru 560100, India

2Department of Zoology, Maltidhari College (A constituent college of Patliputra University, Patna), Naubatpur Patna

Abstract

As a first step towards clarifying the basis of the cooperation and conflict seen in chimeric binary mixes of Dictyostelium giganteum, we examined the karyotype of six natural isolates. All six had 5 haploid chromosomes. No meiotic figures were seen. Fluorescence in-situ hybridization was carried out using conserved D. discoideum centromeric DNA sequences as probes. From it, we infer that two chromosomes are sub-metacentric, one is metacentric and two are telocentric.

Author Contributions
Received 17 Oct 2023; Accepted 11 Apr 2024; Published 20 Jul 2024;

Academic Editor: Amal Ibrahim Hassan Ibrahim, Department of radioactive isotopes.

Checked for plagiarism: Yes

Review by: Single-blind

Copyright ©  2024 Rishikesh Kumar, et al

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Creative Commons License     This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Competing interests

The authors declare no conflict of interest.

Citation:

Rishikesh Kumar, Pooja S. Kulshreshtha, Vidyanand Nanjundiah, Jayarama S. Kadandale (2024) The Chromosomes of Dictyostelium Giganteum. Journal of Chromosomes - 1(1):1-11.

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Introduction

We report on the chromosomal constitution of six naturally-occurring strains of Dictyostelium giganteum. D. giganteum was chosen for two reasons. First, the cytology of this species of Dictyostelid is unkown. Second, our study is motivated by the fact that we have data on social behaviour in pairwise chimaeras made up of different strains of the species 1, 2. The strains in a chimeras differ in their reproductive fitness as measured by the ability to sporulate. That opens up the possibility of identifying correlations between chromosomal or DNA-level variations between strains of a species and relative fitness.

Sorocarpic amoebae, which are found in several major groups, display a remarkable transition from a predatory free-living phase to a cooperative multicellular phase 3. The best-studied among them belong to the Amoebozoa and are known as the Dictyostelid or cellular slime moulds (CSMs) 4. Their life cycle makes the CSMs ideally suited to address questions related to the evolution of sociality with reproductive division of labour5. There are many studies dealing with the evolutionary basis of social behaviour, in particular of so-called altruistic behaviour and cheating, in the CSMs, and they involve both models and experiments 6, 7, 8. However, while one can speculate on what is responsible for the maintenance of sociality in the CSMs, the absence of information on heritable variation within any CSM species limits the extent to which one can think usefully about how it originated. We lack information on intra-species differences in chromosomal makeup, and know very little about finer differences at the level of nucleotide sequences though a beginning is being made with the latter 9, 10. The present study is the first step towards remedying the situation. We proceed to report on a karyotype for D. giganteum. Also, we have carried out fluorescence in situ hybridization of D. giganteum chromosomes using probes derived from conserved centromeric sequences of D. discoideum, and using them, have attempted to classify D. giganteum chromosomes as metacentric or submetacentric and telocentric. A comparison of genomic sequences of the same strains is under way and will be reported elsewhere.

Materials and Methods

Strains

The following six previously described strains of Dictyostelium giganteum were used to make chromosome preparations: 46a3, 46c6, F4, F5, F15 and F16. 46a3 and 46c6 are soil isolates from a 50-ha plot of undisturbed forest in the Mudumalai nature reserve 1, 2. F4, F5, F15 and F16 are derived from different spores in a single fruiting body isolated from elephant dung, also from the same reserve 2, 11. Following their isolation from the wild, the strains were sub-cultured and maintained either in the form of fruiting bodies on non-nutrient agar plates or stored as spores in glycerol at -80oC12. Dictyostelium can be grown in suspension or in culture dishes and either axenically or in the presence of bacteria. Media and buffers required for culturing were prepared according to published guidelines and available in the Dictyostelium Web resource 13. Dictyostelium giganteum was grown with Klebsiella aerogenes on SM agar plates using a modified protocol. A lawn of bacteria was grown first on an SM agar plate by overnight incubation at 37°C. A single spore head taken from a D. giganteum fruiting body was picked with a sterile wire loop and transferred to 10µl of autoclaved MilliQ water, and the suspension was dropped in the centre of an agar plate that had a previously grown lawn of K. aerogenes on it. The plate was incubated at 22°C for 40 hrs. Amoebae that emerged from the deposited spores grew outwards from the centre of the plate. The method ensures that the cells in the centre enter starvation and begin the phase of multicellular development, while cells in the periphery are always in a vegetative stage, therefore are mitotically active and readily available for making chromosome preparations.

Chromosome preparations

Chromosome preparations were carried out for all the six stains by doing independent sampling of each of them (same stock revived from -80°C) at least 3 times to avoid misinterpretation of results caused due to possible cross contamination of the strains.

Treatment with colchicine

The plate with D. giganteum cells was observed under microscope and the central part of growth area which contained cells in developmental phase was cut and removed. The remaining peripheral area with growth which contained mitotically active cells in vegetative phase was treated with 5ml of 400µg/ml colcemid solution (stock solution of 10mg/ml) prepared in 1xKK2 buffer. The culture plate was incubated for 2 hours with colcemid at 22°C and at very low rpm so that growth of the cells on the SM agar plate would not be disturbed or minimally disturbed while simultaneously getting treated with the colchicine.

Cell collection and washing

The cells were collected by purging the cells with 1ml pipetman in a 10ml falcon tube. The washing was done for four times in 1xKK2 buffer for 10 minutes at 1000rpm. Washing with 1xKK2 buffer ensured the removal of the bacterial cells from the suspension. This was followed by incubation of the cells with water at 22°C for 10 minutes (this worked better than the standard 0.56% KCl hypotonic treatment).

Fixation

Amoebae were fixed at least 4 times with a slight modification of the standard protocol (Brody and Williams, 1974) by using a 6:1 ratio of methanol and glacial acetic acid. After fixative washes the pellet was re-suspended in 1 ml of fresh fixative.

Slide preparation and Giemsa staining

Slides for the karyotype study of six strains of D. giganteum were prepared by dropping 120 µl of cell suspension on the slide and then warming the slide on a 37°C warmer for 30min. Giemsa staining (using a stock solution of 2%) was carried out for 4min using Sørensen's phosphate buffer and rinsing the slides 15-20 times in autoclaved MilliQ water.

Microscopy, karyotyping and quantitating chromosome features

Microscopy: Metaphase chromosomes from the six strains of Dictyostelium giganteum were analysed and imaged using OLYMPUS BX51 microscope at 100x magnification and software from Applied Spectral Imaging (ASI)

Karyotyping: An attempt was made to arrange the chromosomes in the form of a karyotype based on the size of the chromosomes.

Estimation of area of individual chromosomes in a metaphase

Twenty well spread metaphases for each of the six strains were selected. Areas of the individual chromosomes in a metaphase were calculated by using the software Image J 14, 15. Sum of area of all chromosomes in a metaphase was calculated. The proportion of the area of the individual chromosome with the sum of area of all the chromosomes in the metaphase was considered as genome proportion of the individual chromosome. This was done for all the 20 metaphases each of the six strains.

Localization of the chromosome centromeres by FISH

Isolation of genomic DNA of Ax-2

For all the molecular studies, Ax-2 strain was used in order to avoid contamination.Genomic DNA was isolated from vegetative stage of Ax-2 strain of D.discoideum with the help of Qiagen genomic DNA isolation Kit. This was used as template DNA for synthesizing centromere specific FISH probes by PCR.

Selection of centromere sequences

The centromere sequences for Dictyostelium discoideum are known in the case of chromosomes 2 and 3, while for chromosome 1, the first 100,000 base pairs include the centromere region 16. To get the most likely centromere sequence in D. giganteum, we carried out FISH with four centromeric sequences of three centromeres from Dictyostelium discoideum which were Chromosome 3 centromere having accession number FJ387222; Chromosome 2 centromere: FJ387223; Chromosome 2 inner centromere: FJ38722415 and Chromosome1 centromere as 1st 100000 bp of chromosome1. These sequences were obtained from Dictybase. They showed conservation of very high level for >800bp in four regions whose positions are mentiond in the table above. The sequence alignment was done by ClustalW at http://www.ebi.ac.uk/. ClustalW analysis of the four centromere sequences of D. discoideum and found four conserved regions of >800bp (Table 1).

Table 1. Table shows ClustalW analysis of 4 centromere sequences of D. discoideum.
4 Centromere ClustalW
Sl. No. Position Size
1 29794-32216 2422bp
2 57279-59137 1858bp
3 52002-53557 1555bp
4 25202-26005 0803bp

Probe preparation

Primers were designed with the help of Gene Runner Software 17, for the four selected conserved regions of centromere sequences of D. discoideum (Table 2). These primers were used to synthesize FISH probes by PCR amplification (with Dig labelled UTP) of the respective sequence according to a published standard protocol 18.

Table 2. Table shows primer sequences designed for FISH probe synthesis with the help of Gene Runner software, for the selected conserved regions of centromere sequences of D. discoideum
Primer from 4 Centromere ClustalW
Sl. No. Product Length Position Sequence Tm %GC Primer Length
1. 2292 29794-32216 FP-CCTGTACTTCGAATGTTGAGAGARP-ATCTACAGTATCGTTTGATTTCCA 63.563.1 43.533.3 2324
2. 1858 57297-59137 FP-GACAACAGCAGAGAAGCCATARP-CCAAGTTACGACTATGTTCTTACA 6261.6 47.637.5 2124
3. 1555 52002-53557 FP-CAGTATTTAAGAAACCACCAGATTRP-ACCAAACAATCAGTAGAGTCGATA 62.562.9 33.537.5 2424
4. 803 25202-26005 FP-TCGGTCAAATACAGATGGATCTRP-CCTAAGGAGTAACAACTGATTCAA 63.263 40.937.5 2224

Fluorescence in situ hybridization for localization of centromeres

FISH was carried out on metaphase chromosome preparation of 46a3 strain of Dictyostelium giganteumbwith the help of a standard protocol used for human chromosomes, with slight modification 19.

In-house hybridization buffer (50% (v/v) formamide, 2× SSC, 10% Denhardt's solution, 0.1 M NaPO4 buffer) as described by 20 without SDS and slight variation of remaining solution componentswas used for preparing probe mix. Post- hybridization washing was carried out at 45°C. Hybridization signals were amplified using Fluorescent Antibody Enhancer set for DIG Detection using the protocol described by the manufacturer (Roche Applied Science, Germany). Figure 1

The FISH signals on the metaphase chromosomes, indicating the possible location of centromeres, were captured and analysed using OLYMPUS BX61 fluorescent microscope at 100x magnification and Applied Spectral Imaging software.

Figure 1.Agarose gel showing products of gradient PCR for the four centromere probes.
Agarose gel showing products of gradient PCR for the four centromere probes.

Results

Modal chromosome number in Dictyostelium giganteum

A minimum of 60 metaphases were analysed for each of the six strains of Dictyostelium giganteum (46a3, 46c6, F4, F5, F15 and F16); representative figures are shown in Figure 2. In all six, the metaphase chromosome number ranged from 4 to 6 with a frequency that was almost the same but differed slightly between them (Table 3). The modal number in each case was five. It appears reasonable to conclude that the haploid chromosome number in these strains of Dictyostelium giganteum, and probably in the species as a whole, is 5. No meiotic figures were seen in any preparation.

Figure 2.Representative metaphase spread of the six strains of D. giganteum confirming the modal number to be 5.
Representative metaphase spread of the six strains of D. giganteum confirming the modal number to be 5.

Table 3. Table shows the frequency of occurrence of cells with different number of chromosomes in six strains of D. giganteum in total for all six strains 1042 metaphases were analysed of which there were 452 for 46a3, 107 for 46c6, 63 for F4, 61 for F5, 222 for F15 and 137 for F16.
Number of Chromosomes Frequency of 6 Strains of Dictyostelium giganteum
46a3 46c6 F4 F5 F15 F16
4 0.05 0.05 0.10 0.18 0.11 0
5 0.87 0.77 0.83 0.77 0.82 0.80
6 0.08 0.10 0.03 0.05 0.07 0.20
>6 0 0.07 0.05 0 0 0

Classification of chromosomes based on size

In all the six strains of D. giganteum analysed in this study, the chromosomes can be classified into 3 groups – two large chromosomes, one medium chromosome and two small chromosomes (Figure 3).

Figure 3.Classification of chromosomes in six strains of Dictyostelium giganteum based on size.
Classification of chromosomes in six strains of Dictyostelium giganteum based on size.

Genome content present in each chromosome in six strains of D. giganteum

Assuming the modal number to be 5 based on the data obtained, 20 well spread metaphases with modal number 5 were selected from each strain. By making use of the software Image J, the area of each chromosome and the proportionate area with respect to the total area of all 5 chromosomes in a metaphase were calculated for each of the 20 metaphases for all the six strains of D.giganteum. This data was used to arrive at a rough estimate of proportion of genomic content of each of the 5 chromosomes in all the six strains (Table 4).

Table 4. Estimate of relative genomic content (expressed as percentages) of the five chromosomes in all six strains of D. giganteum.
StrainsChromosomes 46a3 46c6 F4 F5 F15 F16 Average
1 35.11 34.93 34.82 33.93 34.64 34.19 34.60
2 30.79 30.62 30.83 30.43 30.54 29.37 30.43
3 16.44 17.42 16.44 17.10 16.89 16.95 16.87
4 9.43 9.41 9.5 9.88 9.65 10.52 9.73
5 8.21 7.60 8.38 8.64 8.25 8.95 8.33

Based on the above data it can be concluded that the genomic content of chromosomes 1, 2, 3, 4 and 5 in all strains of D. giganteum is approximately the same, and amounts to 35%, 30%, 17%, 10% and 8% respectively. However, the small differences may be meaningful, and what they might imply will be addressed elsewhere. Figure 4

Figure 4.FISH with centromere probes (representing the 4 conserved sequences) of Dictyostelium discoideum on chromosomes from 46a3 strain of Dictyostelium giganteum, All the four panels a,b,c and d show the FISH done in 46a3 strain of Dictyostelium giganteum with the centromere probe (representing 4 conserved sequences). Numbers indicate the chromosome number based on size. Numbers shown as 1/2 in all the four panels and 4/5 in panel d indicate the ambiguity involved in denoting indicated chromosome exactly as 1 or 2 in all panels and 4 or 5 in panel d. 1/2 and 4/5 should be read as one or two and four or five respectively.
FISH with centromere probes (representing the 4 conserved sequences) of Dictyostelium discoideum on chromosomes from 46a3 strain of Dictyostelium giganteum, All the four panels a,b,c and d show the FISH done in 46a3 strain of Dictyostelium giganteum with the centromere probe (representing 4 conserved sequences). Numbers indicate the chromosome number based on size. Numbers shown as 1/2 in all the four panels and 4/5 in panel d indicate the ambiguity involved in denoting indicated chromosome exactly as 1 or 2 in all panels and 4 or 5 in panel d. 1/2 and 4/5 should be read as one or two and four or five respectively.

Localization of centromere by FISH and classification of chromosomes

Fluorescence in situ hybridization of probes representing conserved centromeric sequences of D. discoideum on the metaphase chromosomes of 46a3 strain of D. giganteum, indicated that two of the large chromosomes are sub metacentric, the medium chromosome is telocentric, one of the small chromosomes is metacentric and the other small chromosome is telocentric.

Discussion

To the best of our knowledge, apart from D. discoideum, chromosome-based studies have not been carried out on any other species of Dictyostelium except the simple karyotyping in Dictyostelium caveatumn21. Our study is also first inter-strain study. For a long time it was believed that D. discoideum had 7 haploid chromosomes 22. Subsequently, pulse-field gel electrophoresis revealed 5 chromosomes 23; the currently accepted number is 6 24, 25, 26. The related species Polysphondyliumviolaceum is said to have 11 or 12 chromosomes 27.

Our study involved chromosome analysis of six distinct strains of D.giganteumand 3 independent samples for each strain, leaving little room for any ambiguity regarding the accuracy of chromosome number arrived at. The haploid chromosome number in all six strains of D. giganteum analysed in our study is found to be 5. The chromosomes can be classified into 3 groups, namely group 1 with two large-sized chromosomes, group 2 with one medium-sized chromosome and group 3 with two small-sized chromosomes. The genome size of D. discoideum is 34.042MB and the genome of an axenic strain has been completely sequenced 25. Preliminary results from work in progress indicate that the size of the D. giganteum nuclear genome is about 32 Mb (average of 6 strains; unpublished data). Based on that, and from the relative sizes of the various chromosomes in D. giganteum, we estimate their genomic contents as 11.07Mb, 9.73Mb, 5.40Mb, 3.11Mb and 2.66 Mb for chromosome Nos. 1,2,3,4, and 5 respectively.

FISH has been used in the past to locate the position of the centromere in human chromosomes 28, 29. Several experiments in past relating to FISH as 30, 31 has shown that DIRS-1 sequences are characteristic of D. discoideum centromeres. But application of FISH to identify centromere has been done by 32 who used this technique to show that Dictyostelium centromeres contain DIRS-1, but their FISH has not been performed in proper metaphase cells. We find some studies on centromere which give the information that chromosomes appear to be acrocentric 33, 34, one class of complex repeat in genome of D. discoideum serve as centromere 25 and sequences which compose the functional chromosomal elements like centromere are not conserved and appear to have underwent several modifications 30, 35. Our study is the first of its kind where we have localised the centromere in the chromosome of D. giganteum using the probe from D. discoideum. Data obtained from our FISH studies has indicated that two of the large chromosomes are sub metacentric, the medium chromosome is telocentric, one of the small chromosomes is metacentric and other small chromosome is telocentric.We have not carried out FISH with other D. giganteum chromosomes, but given the similarity in morphologies, assume that their centromeres will be similarly located.

Conclusions

The modal chromosome number in D. giganteum is five. On basis of size it can be classified into three groups of which first group comprises of two large chromosomes, second group contains a medium chromosome and third group has two small chromosomes. On basis of centromere position its chromosomes of 46a3 strain of D. giganteum can be classified into metacentric, submetacentric and telocentric chromosomes. One of the small chromosome is metacentric, the other small chromosome along with medium sized chromosome is telocentric and two of the large chromosome is submetacentric.

Patents

Not applicable

Supplementary Materials

Not applicable

Author Contributions

For this article contributions done by authors are as follows viz. Conceptualization, V.N.; Methodology, P.K, R.K.; Software, P.K, R.K.; Validation, J.K.; Formal Analysis, R.K., P.K.; Investigation J.K.; Resources, J.K.; Data creation R.K., P.K.; Writing original draft preparation, R.K., P.K., V.N.; Writing review and editing, J.K., V.N.; Visualization, R.K.; Supervision, J.K. and Project administration, J.K. All authors have read and agreed to the published version of the manuscript.

Funding

The research was supported by Internal funding of Centre for Human Genetics, Bangalore.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We are very thankful to Prof. H. Sharat Chandra for his kind support and supervision.

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