Showing papers in "Chromosoma in 1976"

Reverse fluorescent chromosome banding with chromomycin and DAPI.

Abstract: Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.

Light and electron microscopy of rat kangaroo cells in mitosis. III. Patterns of chromosome behavior during prometaphase.

Abstract: Chromosome orientation and behavior during prometaphase of mitosis in PtK1 rat kangaroo cells were investigated by cinemicrography and electron microscopy. The first chromosome movements occur soon after the nuclear envelope begins to break down in the region near each pole. Initial chromosome behavior is primarily determined by the distance from the kinetochore region to the spindle poles. The predominant pattern is a movement to and/or association with the proximal pole. Movement to and association with the more distant pole, or direct alignment at or near the spindle equator (direct congression) are less frequent patterns. Except for rare cases, pole-associated chromosomes congress sooner or later and most congressed chromosomes oscillate about the equator. — Ultrastructural observations suggest that pole-associated chromosomes are oriented only to the proximal pole (monotelic or syntelic orientation) and they demonstrate that the sister-kinetochores of congressing or oscillating chromosomes are oriented to opposite poles (amphitelic orientation). — Based on the structure of the early prometaphase spindle and four assumptions concerning the formation of kinetochore fibers and their force-producing interaction with complementary elements, the different patterns of chromosome behavior observed can be explained as a result of synchronous or asynchronous formation of sister-kinetochore fibers. The few chromosomes whose kinetochore region is approximately equidistant from the poles amphi-orient immediately because their sister-kinetochores form fibers synchronously and they congress directly because of the bidirectional forces to which they are subjected. The kinetochore region of most chromosomes is not equidistant from the poles. Therefore, they form a functional fiber first to the nearer pole and move to, or associate with, it because of the unidirectional force. Eventually, however, these chromosomes achieve amphitelic orientation and congress. Once established, amphitelic orientation is stable. Re-orientations do not occur during congression or oscillatory movements.

Robertsonian metacentrics in the mouse.

Abstract: A survey is given on the occurrence, the geographic origin and the arm composition of 27 Robertsonian fusion metacentric chromosomes of wild populations of the mouse. Their study is of twofold interest: a) it is possible to introduce these naturally occurring metacentrics in laboratory strains for experimental use. At present, altogether 34 metacentric chromosomes of different composition are available including 7 cases of metacentrics known from laboratory strains of the mouse. b) With the search for metacentrics in the mouse and with their identification insights are permitted in the role of Robertsonian changes in the course of mammalian evolution — Several separate populations of the mouse with different sets of multiple (up to 9) metacentrics have been found in Switzerland and Italy. Some of the individual metacentrics may occur in different populations. The participation of an acrocentric autosome in the formation of metacentrics seem to be at random, but the sex chromosomes are never included in a metacentric. — Homology of the arms involved in metacentrics is conserved, so that in meiosis of interpopulation hybrids long chains or rings are observed. They may include up to 16 metacentrics arranged according to the alternating homologies of their arms. — Reduction of fertility of single or multiple metacentric heterozygotes and of the interpopulation hybrids is due to mechanisms of segregational imbalance and subsequent prenatal elimination of fetal offspring, but it follows also the pattern of male limited hybrid sterility. — From an evolutionary view point, karyotype rearrangements of Robertsonian type may initiate reproductive isolation, which prepares the ground for further genetic diversification and, as in the case of the mouse, of incipient speciation.

The meiotic prophase in Bombyx mori females analyzed by three-dimensional reconstructions of synaptonemal complexes

Abstract: Serial sectioning followed by three dimensional reconstruction of lateral components of the synaptonemal complex have been used to follow chromosome pairing during the prophase of the achiasmatic meiotic division in the silkworm, Bombyx mori. During leptotene and early zygotene, the lateral components become attached to the nuclear envelope at a specific region, thus forming a chromosome bouquet. The attachment of lateral components to the nuclear envelope precedes the completion of the components between their attachment points. Synapsis and synaptonemal complex formation start during the period of lateral component organization in the individual nucleus. Telomeric movements on the nuclear envelope occur at two stages of the prophase: the chromosome pairing appears to be initiated by an association of unpaired ends of homologous chromosomes, the nature of this primary attraction and recognition being unknown. Secondly, the paired chromosomes become dispersed in the nucleus by shifting of attachment sites of completed synaptonemal complexes at the end of zygotene. This movement is possibly related to a membrane flow occurring during this stage. Membrane material is synthesized at the region of synaptonemal complex attachment. Later, the excess membrane material is shifted to the opposite pole where it protrudes into the lumen of the nuclei thus forming vacuoles. — Two previously undescribed features of chromosome pairing were revealed. In late zygotene, chromosome pairing and synaptonemal complex formation were frequently observed to be delayed or even prevented over a short distance by interlocking of two bivalents, both being attached to the nuclear envelope. Such interlocking of bivalents was not found in pachytene. Secondly, one nucleus was found in which two homologous chromosomes were totally unpaired while the remaining 27 bivalents were completed or in a progressed state of pairing. The lateral components of the two unpaired chromosomes had the same length and were located several microns apart, thus eliminating the possibility of a permanent association of homologous chromosomes before the onset of meiosis in Bombyx mori females. — During pachytene, one of the 8 cells belonging to the syncytial cell cluster characteristic of oogenesis continues the meiotic prophase whereas the remaining 7 cells, the nurse cells, enter a different developmental sequence, finally resulting in their degeneration. The synaptonemal complex of the oocyte develops into a sausage-like structure after pachytene by a deposition of dense material onto the lateral components, thus filling out most of the central region. The diameter of this modified synaptonemal complex reaches at least 300 nm, as compaired to a pachytene width of approximately 130 nm. Also, the length of synaptonemal complexes increases from 212 μ at zygotene/pachytene to at least 300 μ at the modified pachytene stage. In nurse cells, synaptonemal complexes are shed from the bivalents shortly after pachytene simultaneously with a condensation of the chromatin. These free synaptonemal complex fragments associate and form various aggregates, either more or less normal looking polycomplexes or various complex figures formed by reorganized synaptonemal complex subunits. Later stages have not been included in the present investigation.

Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using a BrdU treatment.

Abstract: Replication times for all important chromosome bands, of both types R and Q (277 structures) are analysed. — The R-bands form a group of structures whose DNA replicates during the early S-phase, while the DNA situated in the Q-bands replicates during the late S-phase. — There may not exist overlapping between replication times of these two types of structures. — The widest R-bands are those which are the earliest to replicate; in general, the most intense Q-bands are those which are the latest to replicate. Especially among these last ones, a certain asynchronism exists between the replication times. Finally the heterochromatin of chromosomes 1, 16 and Y and of the short arms of the acrocentrics could contain two types of DNA which replicate at different times.

The time rate and mechanism of chromosome elimination in Hordeum hybrids

Abstract: Seed development at 20±1° C in continuous light was studied during the first 5 days after pollination in diploid Hordeum vulgare, diploid H. bulbosum and the cross, H. vulgare x H. bulbosum, where H. bulbosum chromosomes were eliminated. Developing seeds were fixed and stained at known intervals after pollination and the embryo sac contents dissected out for cytological examination. — In all cases, the pattern of development was similar to that previously described for the Triticeae. After intraspecific pollination, the rate of endosperm and embryo development was significantly faster in H. vulgare than in H. bulbosum. In hybrid tissues, the rate was intermediate, but often much nearer to that of H. vulgare at first. Elimination of H. bulbosum chromosomes occurred only during endosperm and embryo mitoses. Usually, 0–3 chromosomes were lost at any one division but up to 7 were lost at some. Elimination, which occurred as early as zygotic anaphase, was nearly or quite complete in all dividing cells in both embryo and endosperm after 5 days. The mean number of chromosomes lost per nucleus per nuclear cycle was low at first but rose rapidly and stayed high for about a day in each tissue before falling quickly. The rate of elimination in each tissue was maximal when that tissue first synthesized significant amounts of new cytoplasm (day 2 after pollination in the endosperm and day 3 in the embryo). At mitosis, chromosomes being eliminated differed from others only in failing to congress at metaphase or to reach a pole at anaphase or both. — It is noted that in several widely different examples where either haploids are produced when only hybrids are expected, or where chromosomes of one species are preferentially eliminated from hybrid cells, nucleolar activity was suppressed in chromosomes of the genome which was selectively or preferentially eliminated. Consequently, it is suggested that chromosome elimination in Hordeum hybrids may be caused by a disturbed control of protein metablism in hybrid seeds and perhaps H. bulbosum chromosomes are selectively eliminated because they are less efficient than H. vulgare chromosomes at forming normal attachments to spindle protein.

Characterization of Drosophila heterochromatin. I. Staining and decondensation with Hoechst 33258 and quinacrine

Abstract: A number of preliminary experiments have shown that the fluorescence pattern of Hoechst 33258, as opposed to that of quinacrine, varies with the concentration of dye. The metaphase chromosomes of D. melanogaster, D. simulans, D. virilis, D. texana, D. hydei and D. ezoana have therefore been stained with two concentrations of H 33258 (0.05 and 0.5 μg/ml in phosphate buffer at pH 7) and with a single concentration of quinacrine (0.5% in absolute alcohol). The three fluorescence patterns so obtained were shown to be somewhat different in some of the species and the coincide in others. All three stainings gave an excellent longitudinal differentiation of heterochromatin while euchromatin fluoresced homogeneously. — Living ganglion cells of the six species mentioned above were treated with quinacrine and H 33258. Quinacrine induced a generalized lengthening and swelling of the chromosomes and H 33258 the decondensation of specific heterochromatic regions. — A correlation of the base composition of the satellite DNAs contained in the heterochromatin of the species studied with the relative fluorescence and decondensation patterns showed that: 1) the extremely fluorochrome bright areas and those decondensed are present only in species containing AT rich satellite DNA; 2) the opposite is not true since some AT-rich satellite DNAs are neither fluorochrome bright nor decondensed; 3) there is no good correspondence between Hoechst bright areas and the decondensed ones. — AT richness therefore appears to be a necessary but not sufficient condition both for bright fluorescence and decondensation. Some cytological evidence suggests that similarly AT rich satellite DNA's respond differently in fluorescence and decondensation because they are bound to different chromosomal proteins. — A combination of the results of fluorescence and decondensation revealed at least 14 types of heterochromatin; 4–7 of which are simultaneously present in the same species. Since closely related species (i.e. D. melanogaster and D. simulans; D. virilis and D. texana) show marked differences in the heterochromatic types they contain, it can be suggested that within the genus Drosophila qualitative variations of heterochromatin have played an important role in speciation.

Satellite DNA and evolution of sex chromosomes.

Abstract: The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (ϱ=1.700 g cm−3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster. Assay by in situ hybridization.

Abstract: Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.

Electron microscopy of membrane-free folded chromosomes from Escherichia coli.

Abstract: Membrane-free folded chromosomes were purified from log-phase cultures of Escherichia coli and prepared for electron microscopy by aqueous (Kleinschmidt and Zahn) spreading. The appearance of the chromosomes depended on the salt concentrations in spreading. At certain salt concentration, the chromosomes resembled rosettes, with supercoiled loops of DNA radiating from a central core containing RNA. The rosettes support previous models deduced from physical studies of folded chromosomes. Apparently, cores contain must of the visible RNA, and the organization of the core is linked to the organization of the DNA loops.

Satellite DNA and cytogenetic evolution. DNA quantity, satellite DNA and karyotypic variations in kangaroo rats (genus Dipodomys).

Abstract: The genusDipodomys (kangaroo rats) exhibits major interspecies variations in the proportions of highly reiterated satellite DNA sequences in the genome as well as in the chromosome number and the proportions of uni-armed and bi-armed chromosomes. For nearly all of the approximately 22 species of the genus and several subspecies, liver DNA was distributed in neutral CsCl buoyant density gradients into four fractions: principal DNA (1.698 g/ml), intermediate-density DNA (1.702 g/ml), MS satellite (1.707 g/ml) and HS (heavy) satellites (1.713 g/ml). The total nuclear DNA content of diploid liver cells measured in eleven species by quantitative cytophotometry, ranged from 6.9 to 10.9 pg. These data were correlated with known features of the karyotypes of individual species. The salient findings were: (1) that interspecies variations in diploid chromosome number cluster at 52–54, 60–64 and 70–72 (2) that high total nuclear DNA was associated with high chromosome number, and with relatively large amounts of satellite DNA (3) that a high ratio of HS satellites to intermediate-density DNA was generally correlated with a predominance of metacentric and submetacentric chromosomes (high fundamental number). The relationships of satellite DNA to karyotype structure reveal a new level of hierarchy in the genome that appears capable of exerting global control over environmental adaptation and the evolution of new species. This mechanism is consistent with recent hypotheses that changes in the macro-structure of the genome are more important than point mutations in facilitating the rapid phases of animal evolution.

Pachytene mapping in the female silkworm, Bombyx mori L. (Lepidoptera).

Abstract: Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.

Karyotypic variation in the Australian gekko Phyllodactylus marmoratus (Gray) (Gekkonidae: Reptilia)

Abstract: The gekko Phyllodactylus marmoratus has at least three distinct chromosome races; 2n=36, 2n=36 ZZ/ZW and 2n=34. Specimens from these races are morphologically distinguishable, have a degree of habitat specialization and occur in a defined distribution. The 2n=36 race found in Eastern Australia is the presumed primordial type. The 2n=34 race occurs in Western Australia and is regarded as a fusion derivative. The 2n=36 ZZ/ZW race, which is only found on the Murray River system in Eastern Australia has a heteromorphic sex chromosome system present in the female. Giemsa banding suggests that this heteromorphism is the result of a pericentric inversion.

Telomeric satellite DNA functions in regulating recombination.

Abstract: Molecular and cytogenetical analyses of three sibling species of Australian grasshopper, Atractomorpha australis, A. species-1 and A. similis, resolves one of the long standing problems of highly repeated DNA. In this system satellite DNA functions in regulating the level and position of recombination, irrespective of whether the repeated DNA is located in telomeric or centric regions. — Even though the three species do not differ in their euchromatic genome sizes, their relative DNA contents are 1.00/1.10/ 1.41, the difference in genome size being due solely to visible centric or telomeric blocks of heterochromatin. — Antibiotic analytical and preparative ultracentrifugation, in situ hybridization and renaturation kinetic analyses reveal that a large cryptic satellite of A. similis constitutes the heterochromatic telomeric blocks of nearly all autosomes and that the DNA of this satellite is highly repeated. — Comparison of these grasshopper data with published literature of heterochromatic rearrangements in Drosophila and with heterochromatin distribution and recombination patterns in diploid plant species reveals that in every case heterochromatin is implicated in some form of alteration in the meiotic recombination system.

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells.

Abstract: BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.

Contrasting patterns of DNA sequence arrangement in Apis mellifera (honeybee) and Musca domestica (housefly).

Abstract: We have examined the organization of the repeated and single copy DNA sequences in the genomes of two insects, the honeybee (Apis mellifera) and the housefly (Musca domestica). Analysis of the reassociation kinetics of honeybee DNA fragments 330 and 2,200 nucleotides long shows that approximately 90% of both size fragments is composed entirely of non-repeated sequences. Thus honeybee DNA contains few or no repeated sequences interspersed with nonrepeated sequences at a distance of less than a few thousand nucleotides. On the other hand, the reassociation kinetics of housefly DNA fragments 250 and 2,000 nucleotides long indicates that less than 15% of the longer fragments are composed entirely of single copy sequences. A large fraction of the housefly DNA therefore contains repeated sequences spaced less than a few thousand nucleotides apart. Reassociated repetitive DNA from the housefly was treated with S1 nuclease and sized on agarose A-50. The S1 resistant sequences have a bimodal distribution of lengths. Thirty-three percent is greater than 1,500 nucleotide pairs, and 67% has an average size about 300 nucleotide pairs. The genome of the housefly appears to have at least 70% of its DNA arranged as short repeats interspersed with single copy sequences in a pattern qualitatively similar to that of most eukaryotic genomes.

Myths and mechanisms of meiosis

Abstract: A comparative analysis of the meiotic secquence in a wide variety of organisms indicates there is no convincing evidence that: (1) Premeiotic pairing plays any role in the synapsis of homologues. (2) Heterochromatic association facilitates homologous pairing. (3) Chiasmata ever form within segments which are positively heteropycnotic at zygotenepachytene. (4) Localisation of chiasmata depends on prior localisation of pairing or on the occurrence of euchromatin-heterochromatin boundaries. (5) Prior association of centromeres plays any role in determing co-orientation. (6) Any form of supra-chromosomal organisation exists involving permanent association between the members of a haploid complement, and (7) Unequal progeny ratios recovered from structurally modified Drosophila complements arise as a consequence of distributive pairing.--On the other hand there is good evidence that: (1) Interlocking of bivalents can occur regularly in species with a chiasma frequency sufficiently high to regularly produce ring bivalents and in which the chiasmata are localised to the ends of the bivalent. (2) Some forms of terminal association cannot represent terminalised chiasmata. (3) U-type exchanges present at diplotene result from errors in crossing over. (4) Pairing and chiasma formation are not necessary for coorientation, and (5) at least some types of elastic constrictions present at first metaphase represent extended nucleolar organisers.

Detection of nucleolus organizer regions in chromosomes of human, chimpanzee, gorilla, orangutan and gibbon.

Abstract: Nucleolus organizer regions were detected by the Ag-AS silver method in fixed metaphase chromosomes from human and primates. In the human, silver was deposited in the secondary constriction of a maximum of five pairs of acrocentric chromosomes: 13, 14, 15, 21 and 22. The chimpanzee also had five pairs of acrocentric chromosomes stained, corresponding to human numbers 13, 14, 18, 21 and 22. A gibbon had a single pair of chromosomes with a secondary constriction, which corresponded to the nucleolus organizer region. In each case the Ag-AS method detected the sites which have been shown by in situ hybridization to contain the ribosomal RNA genes. An orangutan had eight pairs of acrocentric chromosomes stained with Ag-AS, probably corresponding to human numbers 13, 14, 15, 18, 21 and 22, plus two others. Two gorillas had silver stain over two pairs of small acrocentric chromosomes and at the telomere of one chromosome 1. The larger gorilla acrocentric chromosomes had no silver stain although they all had secondary constrictions and entered into satellite associations.

Nuclear DNA variation in Lathyrus

Abstract: In the genus Lathyrus the divergence and evolution of species was accompanied by large scale changes in nuclear DNA amount. All the species are diploids with 14 chromosomes so that the DNA changes were the result of amplification or deletion of segments within chromosomes. Our evidence indicates that the quantitative changes involve mainly the repetitive, as distinct from the non-repetitive fraction of the chromosomal DNA and, on a cytological basis, mainly heterochromatin in contrast to euchromatin. There is an element of discontinuity in the distribution of DNA amounts among species which suggests that the DNA variation results from a series of separate, spasmodic events. The discontinuities may be viewed, also, as “steady states” from the standpoint of genetic balance and biological fitness.

BrdU-33258 Hoechst analysis of DNA replication in human lymphocytes with supernumerary or structurally abnormal X chromosomes.

Abstract: BrdU-33258 Hoechst techniques have been used to characterize DNA replication patterns in lymphocytes from human females with supernumerary or structurally abnormal X chromosomes. Fluorescence analysis permits identification of late replicating X chromosomes in a very high proportion of cells and affords a high resolution method for determining the interchange points of X-X and X-autosome translocations. Asynchrony among terminal replication patterns of multiple late replicating X chromosomes within an individual cell can occasionally be demonstrated. The arms of isochromosomes usually exhibit symmetrical fluorescence patterns, with replication terminating in bands Xq21 and Xq23 (predominant pattern) or in bands Xq25 and Xq27 (alternative pattern) in both arms. In the vast majority of lymphocytes containing a balanced X-13 or X-19 translocation, the normal X is late replicating. However, DNA synthesis in the translocation products occasionally appears somewhat delayed relative to that expected for an early replicating X, consistent with possible position effects on replication kinetics.

The nucleolus organizer region of maize (Zea mays L.)

Abstract: Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNA's of the various partially hyperploid plants and appropriate controls were extracted and hybridized with 3H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.

Morphology of transcription units in Drosophila melanogaster

Abstract: We have used an electron microscopic analysis to define and to characterize active transcription units of Drosophila melanogaster. The lengths and spacings of nascent ribonuclear protein (RNP) fibers were determined on embryonic chromatin that was spread using techniques introduced by Miller and Beatty (1969). The data are consistent with the occurrence of specific sites of transcription initiation and termination. We apply the term transcription unit (TU) to a chromatin region bounded by these control sites. Two classes of TUs are active in Drosophila melanogaster embryonic cells--those synthesizing ribosomal RNA and those synthesizing non-ribosomal RNA. The classes can usually be distinguished on the basis of TU size, chromatin morphology and inferred DNA packing ratio, frequency of RNP fibers (number of fibers per mum of chromatin), and the solitary vs. tandem repeat occurrence of fiber arrays. The results indicate that non-ribosomal transcription units have lengths in accord with the expectation that DNA of each chromomere is transcribed as a unit. Some nascent fiber arrays in D. melanogaster have more complex patterns of RNP fiber lengths. We suggest that these are a consequence of cleavage of RNP fibers at specific sites during transcription. These sites of transcriptional control and the amounts of DNA between them provide a basis for further relating units of transcription to units of gene function.

Meiotic drive for B-chromosomes in the primary oocytes of Myrmeleotettix maculatus (Orthopera: Acrididae).

Abstract: Using a modified technique which allowed observation of chromosome orientation in the primary oocyte of grasshoppers at the onset of anaphase, it has been possible to establish that the B-chromosome is distributed preferentially on the egg side of the metaphase plate rather than the polar body side. The frequency of this preferential orientation matches very closely the level of preferential transmission determined from breeding experiments using individuals from the same population. The spindle is asymmetrical in the primary oocyte of this species, and a possible explanation of the meiotic drive is proposed as a result of the conical shape of nucleoplasm surrounding this spindle. The autosomal chiasma frequency of these females is generally lower than comparable males and is increased by the presence of B chromosomes; but the chiasma frequencies of the sexes respond differently to the addition of 1 and 2 B-chromosomes.

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster

Abstract: A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: 1) Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetitive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucleotide long fragments which also contain repetitive sequences.

Characterization of Drosophila heterochromatin. II. C- and N-banding.

Abstract: The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.

Analysis of chromatin-associated fiber arrays.

Abstract: Electron microscopic examination of chromatin from embryonic nuclei ofOncopeltus fasciatus andDrosophila melanogaster reveals arrays of chromatin associated fibers. The lengths and spacings of these fibers were analyzed to provide a basis for defining and interpreting regions of transcriptionally active chromatin. The results of the analysis are consistent with the interpretation of some fibers as nascent RNA with associated protein (RNP). The chromatin segments underlying these fiber arrays were classified as ribosomal or non-ribosomal transcription units according to definitions and criteria described by Foe et al. (1976). — Nascent fibers on active ribosomal transcription units were analyzed and compared forDrosophila melanogaster, Triturus viridescens, andOncopeltus fasciatus. A common feature of the fiber patterns on ribosomal TUs is that origin-distal fibers exhibit greater length variability and a lower slope relative to proximal fibers. The region of increased variability in fiber lengths is correlated with the expected location of 28S ribosomal RNA sequences in the distal half of each ribosomal transcription unit. Because 28S ribosomal RNA appears to contain more extensive regions of base sequence complementarity, we suggest that the length of ribosomal RNP fibers is influenced under our spreading conditions by the secondary structure of the nascent RNA. — In order to calculate the RNA content of RNP fibers, chromatin morphology was used to estimate lengths of transcribed DNA. The packing ratio of DNA in chromatin, which we express as the length of B-structure DNA ÷ length of chromatin, is 1.1.–1.2. and 1.6 for the DNA in active ribosomal and non-ribosomal chromatins, respectively. These DNA packing ratios are used to determine the extent to which nascent RNP fibers are shorter than the transcribed DNA (expressed as DNA/RNP length ratio). For non-ribosomal transcription units and for proximal fibers of ribosomal transcription units, DNA/RNP length ratios are relatively constant within each array. However, considerable variability in this ratio (4–23) is observed for different arrays of fibers. Possible sources of this variability are considered by comparing ratios derived from the presumably identical ribosomal transcription units. — Further analysis of the morphology of nascent fibers may elucidate the contributions of proteins and successive RNA sequences to RNP structure.

Electron microscopy of membrane-associated folded chromosomes of Escherichia coli

Abstract: Membrane-associated folded chromosomes were purified from log-phase cultures of Escherichia coli 15 TAU-bar and prepared for electron microscopy by aqueous spreading techniques. A spectrum of structures was observed, ranging from condensed structures with no DNA fibers visible, to extended structures with DNA fibers. In the extended structures, loops of DNA radiated from residual envelope, the loops sometimes appeared super-coiled, and both their number and apparent contour length approximated previous estimates from physical and biochemical data. It is proposed that the structures with free DNA arose from the condensed structures.

Centromere behavior during interphase and meiotic prophase in Allium fistulosum from 3-D, E.M. reconstruction

Abstract: Centromeres at premeiotic interphase are clustered and situated in a small area of the nucleus opposite to the nuclear envelope associated heterochromatic masses. The centromeres may occur singly or they may associate to form a structure composed of 2 or more centromeres. Many centromere associations are nonhomologous. Interphase centromeres are not attached to the nuclear envelope. — At zygotene and pachytene centromeres are no longer clustered at one pole of the nucleus but rather are distributed throughout the nucleus. Premeiotic associations appear to be resolved prior to meiotic pairing. Only homologous centromere associations occur during zygotene and pachytene. There is no indication that premeiotic centromere associations are involved in prezygotene alignment of homologous chromosomes.

Human Nucleolar Organizer Chromosomes: Satellite Associations

Abstract: The D and G group chromosomes from cultured human lymphocytes exhibit single and multiple satellite associations when stained with silver. Unlike earlier methods this simple and highly repeatable procedure shows physical attachments between satellited regions of various acrocentric autosomes. After studying 1,000 satellite associations from 118 normal individuals, it was found that both single and multiple associations occur with frequencies that correlate with random expectancies.

Single copy DNA and structural gene sequence relationships among four sea urchin species.

Abstract: Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. drobachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.