Unpaired Chromosomes Research Articles

Karyotypic conservation in the mammalian order monotremata (subclass Prototheria).

The order Monotremata, comprising the platypus and two species of echidna (Australian and Nuigini) is the only extant representative of the mammalian subclass Prototheria, which diverged from subclass Theria (marsupials and placental mammals) 150-200 million years ago. The 2n = 63 male, 64 female karyotype (newly described here) of the Nuigini echidna is almost identical in morphology and G-band pattern to that of the Australian echidna, from which it diverged about a million years ago. The karyotype of the platypus (2n = 52) has several features in common with those of the echidna species; six pairs of large autosomes, many pairs of small (but not micro-) chromosomes, and a series of small unpaired chromosomes which form a multivalent at meiosis. Comparison of the G-band patterns of platypus and echidna autosomes reveals considerable homology. Chromomycin banding demonstrates GC-rich heterochromatin at the centromeres of many platypus and echidna chromosomes, and at the nucleolar organizing regions; some of this heterochromatin C-bands weakly in platypus (but not echidna) spreads. Late replication banding patterns resemble G-banding patterns and confirm the homologies between the species. Striking heteromorphism between chromosomes of some of the large autosomal pairs can be accounted for in the echidna by differences in amount of chromomycin-bright, late replicating heterochromatin. The sex chromosomes in all three species also bear striking homology, despite the difference in sex determination mechanism between platypus (XX/XY) and the echidna species (X1X1X2X2/X1X2Y). The platypus X and echidna X1 each represent about 5.8% of haploid chromosome length, and are G-band identical. Y chromosomes are similar between species, and are largely homologous to the X (or X1).

Chromosome 9 of Ellobius lutescens is the X chromosome.

Ellobius lutescens carries an apparently identical karyotype (2n = 17) in both sexes. On the basis of indirect evidence the unpaired chromosome 9 has been considered to represent the X chromosome of this species. We have obtained data to substantiate this view by four different techniques. After fusion of HPRT- RAG cells with E. lutescens fibroblasts we demonstrated that the enzymes HPRT and G6PD are localized on the presumptive X chromosome. By analysis of pachytene figures after silver staining we showed by electron microscopy that the single chromosome exhibits the typical features of an X chromosome in male meiosis. Hybridization of (GATA)4 and (GACA)4 oligonucleotide probes to E. lutescens DNA revealed several distinct bands in the high molecular weight range some of which appeared to be specific for the individual but not for the sex of the animal. Hybridization in situ of the (GATA)4 probe on metaphase spreads of E. lutescens did not highlight any particular chromosome segment but showed a significant deficit of these sequences in chromosome 9. These observations are discussed with respect to their bearing on X chromosome determination. Finally it is concluded that E. lutescens should be an ideal tool for testing candidate genes assumed to be involved in primary sex determination.

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by γ-irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) - all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F1 progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.

The chromosomal location of a gene (msg) affecting megasporogenesis in durum wheat

An aneuploid durum wheat line (Triticum turgidum L. var. durum) having 13 chromosome pairs and 2 unpaired chromosomes at metaphase I of meiosis in pollen mother cells (i.e., monosomic for chromosomes 7A and 7D) was observed to produce some progeny plants with 2n = 40 chromosomes. These aneuploid (triploid) plants were usually weak and sterile. Triploid plants also occurred in the progeny of durum plants monosomic for chromosome 7A, or in progeny of plants that were mono-telodisomic or ditelomonotelosomic for chromosome 7Aq (13 II + 1 t II or 13 II + t II + t I) but not in the progeny of plants ditelomonotelosomic for chromosome 7Ap (13 II + t II + t 1). Therefore, there is a gene(s) on chromosome arm 7Ap that prevents the production of diploid (2n) egg cells in wheat. In the absence of 7Ap, a portion of the egg cells have 26 chromosomes, which when fertilized with a pollen nucleus with 14 chromosomes, produces progeny plants with 2n = 40 chromosomes. The data also indicated that chromosome arm 7Dp probably contains a second gene that is capable of preventing the production of triploid plants. Key words: Triticum turgidum L. var. durum, polyploidy, aneuploid, triploid, monosomic.

Synaptonemal complex analysis in spermatocytes and oocytes of tertiary trisomic Ts(512)31H mice with male sterility.

Whole-mount preparations of silver-stained spermatocytes and oocytes from Ts(512)31H mice were examined in the electron microscope. The 5(12) chromosome was associated with the XY bivalent in the large majority of spermatocytes, whereas in about one-half of the oocytes, the 5(12) was associated with either unpaired chromosomes or heterochromatic parts of chromosomes or showed self-synapsis. There was a tendency for 5(12) chromosomes to be more fully heterochromatic in oocytes than in spermatocytes. A large proportion of oocytes (50%) and a much smaller proportion of spermatocytes exhibited various errors of chromosome pairing, but these proportions were only marginally greater than in control gametocytes from mice with normal karyotypes. It is concluded that the observed errors of pairing bear no simple relation to the almost complete breakdown of spermatogenesis and the marked impairment of oogenesis that occur in tertiary trisomic Ts(5(12))31H mice.

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.

Chromosomes of five species of sea-skater (Gerridae-Heteroptera)

We report here the first chromosome numbers for sea-skaters. Meiotic metaphase figures were obtained from the testes of three littoral species: Asclepios shiranui, Halobates flaviventris and H. robustus and two pelagic species: H. germanus and H. micans. In the four species of Halobates 2nd♂=31, the highest chromosome number so far determined for the Gerridae. In a proposed ancestral form, A. shiranui, 2n♂=23. Males of all five species have an unpaired chromosome, assumed to be the X chromosome of an XO sex chromosome system.

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological, autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after 3H-thymidine and 3H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. 3H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). 3H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.

Ultrastructural characterization of the meiotic prophase A tool in the assessment of radiation damage in man

• The three-dimensional reconstruction of meiotic nuclei from serial sections micrographed in the electron microscope has provided information about man and several other organisms that is not obtainable by light microscopy or biochemical analysis. • At zygotene, the previously unpaired chromosomes align and form synaptonemal complexes between homologous chromosome segments either by progressive initiation from the telomeres or by interstitial recognition. Chromosome and bivalent interlocking at zygotene is a regular phenomenon and occurs at a frequency of 0.7–4.0 per nucleus in samples of meiocytes analyzed from different organisms. This frequency is reduced to 0.1 per nucleus at pachytene. The interlocking are resolved by breakage and precise rejoining of the broken ends. This breakage and rejoining can also occur in the absence of the DNA nicking and repair involved in crossing-over. The synaptonemal complexes combining homologous chromosome segments are stabilized by recombination nodules, after which a second round of synaptonemal complex formation between as yet unpaired or unstably paired chromosome segments occurs, apparently for optimization of bivalent formation. Non-homologous pairing with the synaptonemal complex can take place in this phase of pachytene. • Continuity between recombination nodules and chromatin chiasmata has been traced at the ultrastructural level but not all nodules lead to chiasmata. The distributions of recombination nodules among the bivalents and along the bivalents at successive stages of meiotic prophase show that the nodules are placed at random at early-zygotene after which bivalents without nodules have preference for the acquisition of these structures. Chiasma intereference appears as a consequence of the limited number of recombination nodules available together with a decreased affinity of a bivalent arm with a nodule for additional ones. The relevance of these observations in the study of genetic damage by radiation is discussed.

Hybrids between the tetraploids of Bromus inermis and B. pumpellianus

Tetraploid F1 hybrids from Bromus pumpellianus ssp. dicksonii (2n = 4x = 28) × B. inermis (2n = 4x = 28) were highly sterile. Chromosome pairing was regular but a low frequency of unpaired chromosomes and multivalents at metaphase I and a low frequency of bridge and fragment configurations at anaphase were recorded. These results indicate that the chromosomes of the parents differ by one or more interchanges and one or more small inversions. The karyotype of the F1 hybrid suggested some structural difference in the satellite-bearing chromosomes of the two species but no other definite evidence of structural changes.

INCIPIENT GENOME DIFFERENTIATION IN GOSSYPIUM. III. COMPARISON OF CHROMOSOMES OF G. HIRSUTUM AND ASIATIC DIPLOIDS USING HETEROZYGOUS TRANSLOCATIONS

Hybrids between upland cotton (G. hirsutum, genome constitution 2A(h)D(h)) and either A-genome or D-genome diploid species exhibit 26 paired and 13 unpaired chromosomes at metaphase I. The A(h) and D(h) genomes are therefore considered homoeologous with those of the respective diploids. Previous studies, nevertheless, revealed a low level of ("incipient") differentiation between D(h) and various diploid D genomes. The diploid A genomes have been regarded as more closely homologous to A(h) on the basis of low preferential pairing and autotetraploid segregation ratios in allohexaploids.-The present study addressed the following questions: Are the diploid A genomes differentiated from A(h) in meiotic homology? If so, is the differentiation manifested equally by all 13 chromosomes or is it localized in certain chromosomes?-Three diploid A-genome lines representing G. herbaceum and G. arboreum were hybridized by in ovulo culture of embryos (1) with a standard line of G. hirsutum, which differs from G. herbaceum by two and from G. arboreum by three naturally occurring reciprocal translocations involving chromosomes 1-5, and (2) with six lines homozygous for experimental translocations involving chromosomes 6, 7, 10, 11, 12 and 13. Chiasma frequencies in hybrids were compared with those in appropriate G. hirsutum controls. In every comparison overall chiasma frequencies were slightly lower in the hybrids. Therefore A(h) appears to be differentiated from the diploid A genomes. No localized differentiation was detected in chromosomes marked by experimental translocations. The differentiation may be localized mainly in chromosomes 4 and 5.

Synaptonemal complex formation and distribution of recombination nodules in pachytene trivalents of triploid Coprinus cinereus

Synaptonemal complex formation and distribution of recombination nodules were analyzed in trivalents of triploid Coprinus cinereus. The analysis of 14 completely reconstructed nuclei covering the period from early pachytene to mid diplotene has permitted the following conclusions: at pachytene, chromosome pairing and synaptonemal complex formation were almost exclusively in the form of trivalents. All three homologues were frequently associated at the same site, two central regions combining the three lateral components. The three homologues were never paired with three central regions. The trivalent configurations were maintained unaltered throughtout pachytene and at least the centromeres remained associated at early and mid diplotene. At mid-late pachytene, unpaired chromosome arms were frequently associated with central region material in some cases containing recombination nodules. The free face of such central regions was occasionally bound to an electron dense rod resembling a lateral component. Both central regions of the double complexes in the trivalents were able to bind recombination nodules. An analysis of the distribution of recombination nodules along the trivalents indicated that nodules were distributed independently between the two central regions of the trivalents.

Expermental hybridization within the genus Triturus (Urodela: Salamandridae). III. Evidence for crossing-over, true chiasmata and chomosomal homologies in the spermatogenesis of F1 species hybrids, T. cristatus carnifex female X T. marmoratus male.

Spermatogenesis in the F1 hybrid (2n=24=12 female + 12 male) between the closely related newt species T. cristatus carnifex and T. marmoratus was apparently normal up to pachytene. Many unpaired chromosomes were present at diplotene and a typical diakinesis was lacking. Primary spermatocytes at meta- and meta-anaphase contained up to 12 regular intergenomal bivalents and a corresponding number of univalents when less then 12 II. Most chiasmata were terminal or subterminal, some intercalary. Chiasmata between corresponding heterospecific chromosomes can be reported as true: real crossing over has taken place, proving the presence of primary chromosomal homologies between the 2 sets of the parental species. Evidence for recombination is based on the segregation of particular markers (i.e., subterminal C-bands and NORs) observed in certain chromosomes at metaphase II. One chromatid of single chromosomes can show the T. cristatus "pheno-type" and the other the T. marmoratus phenotype". A few primary spermatocytes contain a certain number of irregular associations (intragenomal or intrahaploid bivalents, irregular intergenomal bivalents, chromosome multivalents) joined by chiasmata which can be defined as anomalous. Other abnormalities concern the occurrence of interlocked bivalents which occasionally show an anomalous exchange between heterologous chromatids. Cytogenetic criteria useful to evaluate the taxonomic relationships between different species have been discussed as well as some possible trends in chromosome evolution and speciation within the genus Triturus.

Experimental Hybridization within the GenusTriturus(Urodela: Salamandridae). II. The Lampbrush Chromosomes of F1Species Hybrids BetweenTriturus Cristatus CarnifexandT. Vulgaris Meridionale

SUMMARYThe lampbrush phase of 6 F1 female hybrids of Triturus cristatus carnifex ♀ × T. vulgaris meridionalis ♂ and 1 female hybrid of the reciprocal cross was analyzed in several growing oocytes. The chromosome constitution of these hybrids was found to be diploid and derived from a regular, although heterospecific amphimixis (2n= n♀ + n♂). All the oocytes contained 24 unpaired lampbrush chromosomes, among which the single univalents of the 2 parental sets were recognized by their cytogenetic markers (loops, spheres, NORs, other objects and lateral projections). Lampbrush univalents can be joined to one another by transitional fusions of spheres and telomeres and, occasionally, by very rare chiasmata which may be defined as anomalous since they form between heterologous chromosomes and probably represent a further impediment to subsequent meiotic stages. Problems concerning chromosomal homologies and cytogenetic aspects of sexuality and sex determination in these species hybrids have been discussed in th...

Chromatin diminution and a chromosomal mechanism of sexual differentiation in Strongyloides papillosus.

Eggs obtained from feces of rabbits infected with Strongyloides papillosus were squashed and the karyotypes were determined. They contained cells with either two long and two medium sized chromosomes (2L2M), or one long, three medium and one short chromosome (L3MS). Two types of parasitic female gonad could be distinguished on the basis of oocyte chromosome morphology at prometaphase of the maturation division. All the oocytes in a gonad contained either two upaired long chromosomes and two unpaired medium sized chromosomes, or two unpaired medium sized chromosomes and two unpaired chromosomes segmented into beads in one region. At the maturation division in mitotic parthenogenesis the beads appear to be lost from one of the chromosomes. This generates a medium sized and a shorter chromosome, which together with the undiminished chromosomes make up the L3MS karyotype. Animals with beaded oocyte chromosomes lay eggs that develop into males. It is suggested that males are heteromorphic for the long homologue due to chromatin diminution, that occurs in the maturation division of mitotic parthenogenesis.

The synaptinemal complex in Rhoeo spathacea.

The synaptinemal complex in meiocytes of Rhoeo spathacea is described. Unpaired zygotene chromosomes do not exhibit well defined axial cores under the ordinary fixations of electron microscopy and appear diffuse. However, the axial core is defined by ethanolic phosphotungstic acid (PTA) although it does not respond to uranyl-EDTA-lead. Thus the core appears to contain histone but not RNA and presents a condition which is modified later in pairing when lateral elements of the synaptinemal complex respond positively to both tests. The total number of attachments of synaptinemal complexes to the nuclear envelope was determined in several nuclei from serial sections. Eleven of the twelve possible attachments were found in one nucleus. It thus seems certain that all must be so attached. In the same manner all chromosomes can be seen to have an attachment to a chromocentre. Chromocentres are often very large and compound in that two kinds of heterochromatin can be distinguished. These states of chromatin within the chromocentre are considered to be a function of the degree of condensation. Segments of synaptinemal complexes are distributed randomly through sections of pachytene nuclei and long uncoiled segments of complexes are frequently found in or near the centres of median nuclear sections. Synaptinemal complexes are also found in chromocentres. Our findings suggest that on completion of pairing, which begins distally, homologous chromosomes in Rhoeo are paired throughout their entire lengths, rather than in small terminal segments only.

DNA Content of Seven Species of Astereae and its Significance to Theories of Chromosome Evolution in the Tribe

Relative amounts of nuclear DNA were determined in root tip cells of seven species of Astereae: Aster hydrophilus Greene, A. oblongifolius Nutt., A. riparius H.B.K., Machaeranthera boltoniae (Greene) Turner and Home, M. brevilingulata (Sch-Bip.) Turner and Home, M. parviflora Gray, and M. tenuis (S. Wats.) Turner and Home. The results show that A. hydrophilus and M. brevilingulata, with a chromosome number of n = 9, have less nuclear DNA than other closely related species which are either n = 4 or n = 5. Cytological analyses of meiosis in the intergeneric hybrid M. parviflora X A. hydrophilus showed cells with two or more small chromosomes of the latter species pairing with single large chromosomes of the former. Pachytene cells of the hybrids M. parviflora X A. hydrophilus, M. parviflora X A. riparius, and M. boltoniae X M. tenuis showed some unpaired chromosome segments. The significance of these results to chromosome evolution in the tribe Astereae is discussed.

DNA CONTENT OF SEVEN SPECIES OF ASTEREAE AND ITS SIGNIFICANCE TO THEORIES OF CHROMOSOME EVOLUTION IN THE TRIBE

Relative amounts of nuclear DNA were determined in root tip cells of seven species of Astereae: Aster hydrophilus Greene, A. oblongifolius Nutt., A. riparius H.B.K., Machaeranthera boltoniae (Greene) Turner and Home, M. brevilingulata (Sch‐Bip.) Turner and Home, M. parviflora Gray, and M. tenuis (S. Wats.) Turner and Home. The results show that A. hydrophilus and M. brevilingulata, with a chromosome number of n = 9, have less nuclear DNA than other closely related species which are either n = 4 or n = 5. Cytological analyses of meiosis in the intergeneric hybrid M. parviflora X A. hydrophilus showed cells with two or more small chromosomes of the latter species pairing with single large chromosomes of the former. Pachytene cells of the hybrids M. parviflora X A. hydrophilus, M. parviflora X A. riparius, and M. boltoniae X M. tenuis showed some unpaired chromosome segments. The significance of these results to chromosome evolution in the tribe Astereae is discussed.

Cytogenetics and Breeding Behavior of a Hexaploid Agrotricum Immune from Wheat Streak Mosaic Virus1

A breeding line of spring wheat, C.I. 15092, selected from Triticum aestivum L. em Thell./Agropyron intermedium (Host) Beauv. bred true for immunity from wheat streak mosaic virus (WSMV). F1 hybrids of C.I. 15092/T. aestivum ‘Fletcher’ had 20 paired and 2 unpaired chromosomes of unequal size at metaphase I of meiosis in pollen mother cells. Apparently C.I. 15092 had one Agropyron chromosome pair carrying genes for immunity from WSMV substituted for a homoeologous wheat chromosome pair. Immunity from WSMV was completely dominant in hybrids. Inheritance data indicated that the Agropyron chromosome was transmitted through 27% male and 21% female gametes of the F1 hybrid C.I. 15092/Fletcher. A small number of immune F2 and F3 plants were comparable to control plants in seed set, indicating that the whole Agropyron chromosome added to the wheat complement was not strikingly harmful in some plants, at least, not to self‐fertility.