A chromosome rearrangement in Neuvospora that produces viable progeny containing two nucleolus organizers

In rearrangement T(VLIVL)AR33 the segment of chromosome 2 bearing the nucleolus organizer is translocated to the end of chromosome 4. When AR33 is crossed by Normal sequence (N), one third of the viable progeny contain a stable nontandem duplication with two organizers per nucleus. The organizer-deficient complementary products are inviable. Chromosomes and nucleoli have been examined during meiosis and postmeiotic nuclear divisions in the ascus, comparing heterozygous AR33 × N crosses with N × N and with crosses heterozygous for other interchanges. When AR33 is heterozygous, asci are of three types having the nucleolus organizer duplicated in 0, 1 or 2 of the meiotic products. Frequencies of the ascus types are as expected from the known positions of rearrangement break points. Nucleoli formed by two organizers frequently fuse. Deficiency nuclei that contain no nucleolus organizer may form one or more small nucleolus-like bodies.     

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation X Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered.     

An insertional translocation in neurospora that generates duplications heterozygous for mating type

In strain T(I-->II)39311 a long interstitial segment is transposed from IL to IIR, where it is inserted in reversed order with respect to the centromere. In crosses of T x T essentially all asci have eight viable, black spores, and all progeny are phenotypically normal. When T(I-->II)39311 is crossed by Normal sequence (N), the expected duplication class is viable while the corresponding deficiency is lethal; 44% of the asci have 8 Black (viable) spores and 0 White (inviable) spores, 41% have 4 Black: 4 White, and 10% have 6 Black: 2 White. These are the ascus types expected from normal centromere disjunction without crossing over (8B:0W and 4B:4W equally probable), and with crossing over between centromere and break point (6B:2W). On germination, 8B:0W asci give rise to only parental types-4 T and 4 N; 4B:4W asci usually give four duplication (Dup) progeny; and 6B:2W asci usually give 2 T, 2 N, 2 Dup. Thus one third of all viable, black ascospores contain duplications.-Recessive markers in the donor chromosome which contributes the translocated segment can be mapped by duplication coverage. Ratios of 2 Dominant: 1 Recessive vs. 1 Dominant: 2 Recessive distinguish location in or outside the transposed segment. Eleven loci including mating type have been shown to lie within the segment, and markers at four loci have been transferred into the segment by meiotic recombination. The frequency of marker transfer indicates that the inserted segment usually pairs with its homologue. Ascus types that would result from single exchanges within the insertion are infrequent, as expected if asci containing dicentric bridges usually do not survive.-Duplication ascospores germinate to produce distinctive inhibited colonies. Later these "escape" to grow like wild type, and genes that were initially heterozygous in the duplication segregate when escape occurs. As with duplications from pericentric inversion In(IL-->IR)H4250 (Newmeyer and Taylor 1967), the initial inhibition is attributed to mating-type heterozygosity, and escape to a somatic event that makes mating type homoor hemizygous.-Twenty additional duplication-generating Neurospora rearrangements are listed and described briefly in an Appendix.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Magnification of rRNA gene number in a Neurospora crassa strain with a partial deletion of the nucleolus organizer

Some progeny from crosses between the Neurospora crassa translocation strain T(IL VL)OY321 and normal sequence N. crassa strains are duplication strains with a partial deletion of the nucleolus organizer. Despite the deletion, these progeny are viable and produce a functional nucleolus. Quantification of rRNA gene number in these deletion progeny demonstrated a significant loss of rRNA genes, down to 60% of the parental wild-type level. Initially, all of these reduced nucleolus organizer (RNO) strains demonstrated a reduction in the rate of mycelial elongation in growth tubes. After several vegetative growth cycles some progeny reverted to the normal growth phenotype, and also showed an increase in the number of rRNA genes to approximately that of the wild type.     

The Mutation SK(ad-3A) Cancels the Dominance of ad-3A+ over ad-3A in the Ascus of Neurospora

A newly induced mutant of Neurospora, when crossed with an ad-3A mutant, produces asci with four viable black and four inviable white ascospores. The survivors always contain the new mutant allele, never ad-3A. The new allele, which is called SK(ad-3A) (for spore killer of ad-3A), is located at or very near the ad-3A locus.--In crosses homozygous for ad-3A, each ascus contains only inviable white ascospores. This defect in ascospore maturation is complemented by the wild-type allele, ad-3A+ (crosses heterozygous for ad-3A and ad-3A+ produce mainly viable ascospores), but it is not complemented by the new SK(ad-3A) allele (all ad-3A ascospores from crosses heterozygous for SK(ad-3A) and ad-3A are white and inviable). In crosses homozygous for SK(ad-3A) or heterozygous for SK(ad-3A) and ad-3A+, each ascus contains only viable black ascospores. SK(ad-3A) does not require adenine for growth, and forced heterokaryons between SK(ad-3A) and ad-3A grow at wild-type rates and produce conidia of both genotypes with approximately equal frequency. Thus, the action of SK(ad-3A) is apparently restricted to ascospore formation. Possible mechanisms of the action of this new allele are discussed.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes

Aldehyde fixation followed by staining with phosphotungstic acid produces differential contrast between the synaptonemal complex and the chromatin of maize pachytene bivalents. Centromeres, heterochromatic knobs and large chromomeres are easily recognised. With this and other staining techniques the nucleolus organizer region can be differentiated into two components. — Microsporocyte nuclei at pachytene were serially sectioned and all ten bivalents reconstructed in five nuclei. An idiogram was derived from the mean chromosome (= synaptonemal complex) lengths, the arm ratios, positions of knobs and the nucleolus organizer region. The idiogram agrees well with that published from light microscopic analyses. However, bivalent lengths are only two thirds of those observed by light microscopy of squash preparations. Many telomeres of the bivalents are connected via chromatin to the nuclear envelope, but a varying number of free bivalent ends are observed in all five reconstructed nuclei. — Bivalents heterozygous for inversion 3b were reconstructed. In the presence of abnormal chromosome 10 (K10) the lateral components of the synaptonemal complex of chromosome 3 formed a typical inversion loop, while in one of the nuclei having no K10 the two lateral components of the long arms of chromosome 3 remained unpaired in the region of inversion heterozygosity. The presence of K10, which increases crossing-over frequencies and promotes intimate pairing at the light microscopic level, was thus found to permit formation of complete synaptonemal complexes in the inverted region. The extra terminal portion of the K10 chromosome folded back on itself and formed a morphologically normal synaptonemal complex in this — possibly non-homologously paired — region. The chromatin of centromeres and knobs from different bivalents were sometimes found to fuse, but the synaptonemal complexes transversing the fused centromeres or knobs retained their individuality.     

Analysis of Nondisjunction Induced by the R-X1 Deficiency during Microsporogenesis in Zea Mays L

The r-X1 deficiency in maize induces nondisjunction at the second mitotic division during embryo sac formation. However, it was not known if this deficiency also induces nondisjunction during the microspore divisions. Microsporogenesis in plants lacking or containing this deficiency was compared using two approaches. First, chromosome numbers were determined in generative nuclei. Many (8.3%) of the generative nuclei in r-X1-containing plants were aneuploid; however, those from control plants were all haploid. Thus, this deficiency induces nondisjunction during the first microspore division. Second, nucleoli were analyzed in microspores. The only nucleolar organizing region in maize is on chromosome 6. If chromosome 6 underwent nondisjunction during the first microspore division, one nucleus in binucleate microspores would contain no nucleolus and the other would contain two nucleoli (or one nucleolus if the nucleoli fused). Only one (0.03%) microspore of this type was observed in control plants while 1.12% were found in r-X1-containing plants. Thus, the r-X1 deficiency induces nondisjunction of chromosome 6 during the first microspore division. However, both of the sperm nuclei in trinucleate microspores contained one nucleolus in r-X1-containing and control plants; thus, this deficiency does not induce nondisjunction of chromosome 6 (and presumably other chromosomes) during the second microspore division.     

Nucleolar organizers and fibrillar centres in Triticum aestivum L., cv. Chinese spring

Serial sectioning of wheat roots prepared for electron microscopy was used to count the number of fibrillar centres per nucleus and nucleolus and to calculate their sizes. After growth at 35 ° C for two days the nucleoli became segregated and a one to one relationship was evident between chromosomal nucleolar organizers and fibrillar centres. This was confirmed using an aneuploid line carrying an additional pair of organizers. Quantitative studies showed that the fibrillar centres occupied a volume 0.24% of the total chromatin reticulum. From this it was calculated that only about 1/3 of the fibrillar centre material was likely to be nucleolar organizer chromatin. The other material was considered to be the protein revealed in silver staining studies. The importance of this was shown by its constant ratio to the size of all nucleoli in a given nucleus. — Evidence was found for the movement and fusion of organizer flanking regions during growth at 35 ° C. The number of junctions between chromatin and nucleolar organizers drops by about half giving one per organizer after segregation, and serial sectioning demonstrated such junctions in close proximity, an arrangement suggestive of incipient fusion.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

Summary The silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.     

Polycoccum crespoae sp. nov., the first report of a lichenicolous fungus on Chondropsis semiviridis (Parmeliaceae)

Abstract:Polycoccum crespoae sp. nov. is described from thalli of Chondropsis semiviridis collected in Australia (Victoria). It has consistently 4-spored asci but differs from known 4-spored species in the size and shape of the ascospores. Attention is also drawn to another undescribed Polycoccum found on another genus ofParmeliaceae , Karoowia adhaerens, from South Africa (Cape Province).     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

The Manifestation of Chromosome Rearrangements in Unordered Asci of Neurospora

Rapid, effective techniques have been developed for detecting and characterizing chromosome aberrations in Neurospora by visual inspection of ascospores and asci. Rearrangements that are detectable by the presence of deficient, nonblack ascospores in test crosses make up 5 to 10% of survivors after UV doses giving 10-55% survival. Over 135 rearrangements have been diagnosed by classifying unordered asci according to numbers of defective spores. (These include 15 originally identified or analyzed by other workers.) About 100 reciprocal translocations (RT''s) have been confirmed and mapped genetically, involving all combinations of the seven chromosomes. Thirty-three other rearrangements generate viable nontandem duplications in meiosis. These consist of insertional translocations (IT''s) (15 confirmed), and of rearrangements that involve a chromosome tip (10 translocations and 3 pericentric inversions). No inversion has been found that does not include the centromere. A reciprocal translocation was found within one population in nature. When pairs of RT''s that involve the same two chromosome arms were intercrossed, viable duplications were produced if the breakpoints overlapped in such a way that pairing resembled that of insertional translocations (27 combinations).—The rapid analytical technique depends on the following. Deficiency ascospores are usually nonblack (W: "white") and inviable, while nondeficient ascospores, even those that include duplications, are black (B) and viable. Thus RT''s typically produce 50% black spores, and IT''s 75% black. Asci are shot spontaneously from ripe perithecia, and can be collected in large numbers as groups of eight ascospores representing unordered tetrads, which fall into five classes: 8B:0W; 6B:2W, 4B:4W, 2B:6B, 0B:8W. In isosequential crosses, 90-95% of tetrads are 8:0. When a rearrangement is heterozygous, the frequencies of tetrad classes are diagnostic of the type of rearrangement, and provide information also on the positions of break points. With RT''s, 8:0 (alternate centromere segregation) = 0:8 (adjacent-1), 4:4''s require interstitial crossing over in a centromere-break point interval, and no 6:2''s or 2:6''s are expected. With IT''s, duplications are viable, 8:0 = 4:4, 6:2''s are from interstitial crossing over, 0:8''s or 2:6''s are rare. Tetrads from RT''s that involve a chromosome tip resemble those from IT''s, as do tetrads from intercrosses between partially overlapping RT''s that involve identical chromosome arms.—Because viable duplications and other aneuploid derivatives regularly occur among the offspring of rearrangements such as insertional translocations, care must be taken in selecting stocks, and original strains should be kept for reference.     

A Mutant Affecting Meiosis in Neurospora

Many mutants affecting meiosis increase the occurrence of aneuploid meiotic products. In Neurospora, mutants of this type cause ascospore abortion which is reflected by an increase in the proportion of ascospores failing to develop black pigment. The usefulness of the criterion white-ascospore-production as a signal for the presence of a mutant affecting meiosis is demonstrated by the recovery of several such mutants. One of these is mei-1 (meiotic-1), a recessive mutant on linkage group IV. Crosses homozygous for mei-1 produce 90% white ascospores (vs. 5% in wild-type crosses). Viable ascospores, invariably black, are always disomic for one or more linkage groups; the chromatids assorted into viable ascospores do not engage in crossing over in meiosis. The distribution of viable ascospores in individual asci suggests that all meioses are defective in the first meiotic division, and that most meioses are defective in both divisions.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.