Radiation-induced chromosomal rearrangement as an aid to analysis of the genetic constitution of Phaffia rhodozyma

Abstract

Electrophoretic karyotypes of 80 auxotrophic and morphological mutants obtained from two Phaffia rhodozyma strains (ATCC 24203 and ATCC 24229) by γ-radiation were investigated. Contour-clamped homogeneous gel electrophoresis separation of the chromosomal size DNAs revealed 29 new chromosomal patterns after mutagen treatment. No correlation was found between a given type of chromosomal aberration and any phenotypic character. However, analysis of the chromosomal rearrangements proved to be useful for a more exact determination of chromosome number and genome size. The total genome size of ATCC 24229 was found to be 19.3 Mb, with nine chromosomes, while analysis of the mutant derivatives of ATCC 24203 suggested the presence of 11 chromosomes, with an estimated total genome size of 22.2 Mb. The advantages of the analysis of mutant electrophoretic karyotypes for genome characterization are discussed.

Chromosomal rearrangement, Contour-clamped homogeneous gel electrophoresis, Electrophoretic karyotype, Mutagenesis, Phaffia rhodozyma, γ-Irradiation

1 Introduction

Phaffia rhodozyma is the only known yeast species that produces astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) in considerable quantity (200–300 μg/g dry yeast)[1]. This carotenoid pigment could be used as an feed ingredient for salmonid fish produced by aquaculture. The excellent antioxidant properties of astaxanthin are also attractive commercially. Though all-trans astaxanthin can be manufactured synthetically, the demand on the available natural sources has increased[2].

Little is known concerning the genetic make-up of P. rhodozyma. Genetic analysis has been hampered by the lack of a known sexual cycle and the unknown nuclear genome. However, in the past few years, a number of important results have been obtained: the teleomorphic state (Xanthophyllomyces dendrorhous) of P. rhodozyma has been described[3], electrophoretic karyotypes have been established for several wild-type strains [4, 5] and data have been reported on the genetic polymorphisms of this species[6]. Additionally, efficient protocols have been devised for the isolation of different types of mutants from P. rhodozyma[7].

Pulsed field gel electrophoresis (PFGE) has been used successfully in several cases to analyze both the chromosomal organization of a fungal species[8] and the processes (e.g. chromosomal breakage) which affect a given chromosomal constitution[9]. In the present study, the contour-clamped homogeneous electric field (CHEF) technique was used to acquire data relating to (a) the chromosomal rearrangements induced by the ionizing radiation used for mutagenesis, and (b) the exact number and size of the chromosomes of the investigated wild-type P. rhodozyma strains.

2 Materials and methods

2.1 Yeast strains and mutagenesis

Eighty mutants obtained from two wild-type P. rhodozyma strains (ATCC 24203 and ATCC 24229) were investigated. They were isolated after γ-irradiation from a ⁶⁰Co radiation source (1241 Gy h⁻¹). The duration of irradiation was 3.4 h and 3.5 h for ATCC 24203 and ATCC 24229, respectively. Auxotrophic mutants were isolated by means of a selective enzymatic enrichment method described recently[7]. Morphological (color) mutants were isolated from solid YMPG medium[7], after incubation for 7 days following plating of the mutagenized cell population. Individual colonies were subcultured several times to obtain genetically homogeneous isolates.

2.2 Chromosomal sample preparation

Protoplasts were formed as described earlier[4]. Freshly prepared protoplasts were washed three times with 0.7 M KCl, and suspensions of 4×10⁸ protoplasts ml⁻¹ were then mixed with an equal volume of 1.3% low gelling temperature agarose (Sigma, Type VII) in 0.7 M KCl. Blocks were cast, allowed to solidify at 4°C for 5 min, and transferred into 10 ml of incubation buffer (proteinase K, 1 mg ml⁻¹; Tris, 0.01 M; sodium laurylsarcosine, 1%; EDTA, 0.5 M, pH 9.5). Following incubation at 50°C for 24 h, the sample plugs were washed twice in 0.05 M EDTA and stored in the same solution at 4°C until further use.

2.3 Conditions for CHEF

Separations were carried out with a CHEF DR-II electrophoresis system (Bio-Rad). Gels were cast from chromosomal-grade agarose (0.8%; Bio-Rad) in 0.5×TBE (Tris-borate, 45 mM; EDTA, 50 mM, pH 8). The running buffer was 0.5×TBE, continuously circulated at 10°C. Electrophoretic conditions for CHEF: 125 V for 72 h with a switching time of 450 s, and 125 V for 20 h with a switching time of 250 s. Gels were stained for 30 min in 0.5 mg ml⁻¹ ethidium bromide.

3 Results and discussion

The recent developments in pulsed field gel electrophoresis allowed an insight into the previously unknown genetic system of the red-pigmented yeast P. rhodozyma. The investigated wild-type P. rhodozyma strains revealed high chromosomal length polymorphisms; all of them had different electrophoretic karyotypes [4, 5]. The 80 auxotrophic and color mutants isolated from two of these strains (ATCC 24203 and ATCC 24229) are listed in Table 1. In 29 mutants, the electrophoretic chromosomal patterns obtained by CHEF were found to be identical to those of the original (wild) types, but in the other cases (51 mutants) changes in the chromosome number and/or size were detected (Fig. 1).

1

Auxotrophs and color markers in mutants from two P. rhodozyma strains

Strain	Auxotrophic marker	Number of color mutants
		white	yellow	othersa
ATCC24203	lys−	1, 3, 7, 11, 12, 13, 16, 18, 23	4, 8, 10, 19, 22, 25	2, 5, 6, 9, 14, 15, 17, 20, 21, 24
	arg−	26, 28, 33, 36	27, 34, 35	29, 30, 31, 32
ATCC24229	arg−	52, 53, 60, 67, 69	43	37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 68
	met−	70, 72, 73, 74, 75, 76, 78, 79		71, 77, 80, 81

^aDark-orange, pink, dark-pink.

1

Examples of electrophoretic karyotypes of mutant derivatives of P. rhodozyma ATCC 24229 arg⁻ obtained after γ-irradiation. Lanes 1–12: strains 39, 40, 41, 42, 45, 46, 47, 48, 49, 50, 51 and 54. Lane 13: P. rhodozyma ATCC 24229. Mutants 39, 40, 42, 46 and 50 exhibited altered chromosomal banding patterns. All these mutants possess the original dark-pink coloration.

Sixteen new electrophoretic banding patterns were observed for ATCC 24229 mutant derivatives, and 11 for those of ATCC 24203. The latter exhibited many more chromosomal rearrangements (97% of the 36 mutants have new chromosomal patterns) than those isolated from ATCC 24229 (36% of the 44 mutants have new patterns). The different electrophoretic chromosomal patterns observed in these mutants are depicted in Fig. 2Fig. 3.

2

Diagrammatic representation of the new chromosomal banding patterns obtained for ATCC 24229 arg⁻ and met⁻ mutant clones.

3

Diagrammatic representation of the new chromosomal banding patterns obtained for ATCC 24203 lys⁻ and arg⁻ mutant clones.

For demonstration of the stability of the observed chromosome rearrangements, 10 mutant strains were randomly selected, subcultured several times from individual colonies and karyotyped again. Under these experimental conditions, the electrophoretic karyotypes of the progeny clones did not display any difference relative to the original ones (results not shown).

The investigated 80 strains included both auxotrophic and morphological (color) mutants. No correlation was found between a given type of chromosomal aberration and any of the phenotypic characters investigated.

Chromosomal rearrangements have been observed and well documented in a variety of microscopic fungi. Spontaneous morphological mutants of Candida albicans have been found to exhibit abnormal electrophoretic karyotypes[10]. Similarly, a variety of induced [9, 11] chromosomal rearrangements have been described for Saccharomyces cerevisiae. In all of these cases, the investigations provided an efficient means for the analysis of the genetic constituents of a given species.

Though numerous mutant strains with various genetic markers were isolated and characterized from P. rhodozyma, the CHEF analysis of the 80 mutant strains randomly selected from them did not reveal any reproducible and specific set of chromosome rearrangements which exhibit a correlation with a definite phenotypic character. However, these data proved useful in facilitating a more precise determination of the genome size for the wild-type parental strains.

Bands staining more intensely for an electrophoretic karyotype are usually counted as doublets. However, there are some reservations about the mechanical application of this approach[12], and the exact number of chromosomes and the total genome size are therefore difficult to determine. The genome size of ATCC 24229 was previously estimated to be 15.8 Mb[4]. Seven different chromosomal mobility groups, one of which (2.60 Mb) was assumed to be a doublet, were observed (a set of eight chromosomes). Analysis of mutant derivatives reinforced this assumption, but suggested that the 3.5 Mb chromosomal band should also be regarded as a doublet. The basis of this correction was the appearance of stable, reproducible extra bands in certain morphological mutants. Six of the newly detected 16 karyotypes contained one extra chromosomal band (altogether eight), while another six karyotypes revealed two extra bands (altogether nine) in a comparison of the parental strains (ATCC 24229 arg⁻ and ATCC 24229 met⁻). More than nine chromosomal bands were not detected in any of the cases (Fig. 2). This observation and the size of the new chromosomal bands clearly indicate that the 3.5 and 2.6 Mb chromosomal bands represent double, comigrating chromosomal DNAs. It is difficult to assess with any degree of certainty whether these chromosomal DNA species are genetically distinct or homologous.

In a similar analysis, the highest number of reproducible observed stable chromosomal mobility groups in morphological mutants of ATCC 24203 lys⁻ and arg⁻ auxotrophs was found to be 11. There is as yet no satisfactory answer as to why this strain displays a much greater chromosomal rearrangement in the investigated progeny than ATCC 24229. One possible explanation is that the ploidy states of these original parental strains are different; the occurrence of genetically equal chromosomes in one of them might allow the accumulation of more chromosomal aberrations in the cells surviving after mutagenesis. The somewhat larger genome size of ATCC 24203 also supports this assumption. The total genome size of ATCC 24229 was estimated on the basis of the presented results to be 19.3 Mb (nine chromosomes). A similar analysis of the mutant derivatives of ATCC 24203 suggested the presence of 11 chromosomes, with an estimated total genome size of 22.2 Mb; only the 2.9 Mb chromosomal mobility group is assumed to be a doublet.

The comigration of chromosomal size DNAs is an appreciable problem when electrophoretic karyotypes of different fungal species are to be determined. Neglect of this phenomenon could lead to an underestimation of both the chromosome number and the total genome size, while the opposite situation, i.e. presumption of the existence of ‘extra’ chromosomes merely on the basis of differences in relative fluorescence, involves uncertain hypotheses. The application of rare-cutting restriction endonucleases (e.g. Not I)[13], followed by scoring of the generated fragments is also an inaccurate possibility: the vast number of restriction fragments does not allow a precise determination of the sizes of individual chromosomes. Variations of this approach, e.g. the generation of artificial restriction sites for extremely rare-cutting enzymes (e.g. meganuclease I-Sce I)[14], also have limitations: they are complex and expensive procedures. The approach described here does not require a knowledge of the mechanisms of the observed chromosomal rearrangements. Its effectiveness might promote determination of the exact chromosomal number in a variety of fungal species.

Acknowledgements

This work was supported in part by Hungarian Scientific Research Fund (OTKA) Grants F/4 017677 and F 021242.