The structure and behavior of the nucleolus organizers in mammalian cells

The regularly occurring secondary constrictions on metaphase chromosomes of mammalian cells prove to be nucleolus organizers as expected. The expression of nucleolus organizers as secondary constrictions, however, varies from cell to cell and from tissue to tissue, including cultivation in vitro. Electron micrographs of the organizer region show that the nucleolus organizer at metaphase is not a constriction. The width of the organizer area is the same as the condensed chromosomal arms; but the filaments, which are the major components of this region, show a diameter of 50–70 Å. The condensed chromosome arms consist of filaments 150–200 Å in diameter. In some mammalian species, structures similar to the nucleolus organizer are located at the end of chromosomes. These may be terminal nucleolus organizers.Supported in part by Research Grants DRG-269 from Damon Runyon Memorial Fund for Cancer Research, E-286 from American Cancer Society, and HD-2590 from National Institutes of Health.     

The fine structure of the kinetochore of a mammalian cell in vitro

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 Å in diameter surrounded hy a less dense zone 200–600 Å wide. The dense core consists of a pair of axial fibrils 50–80 Å in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oöcytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.The authors also wish to thank Dr. Arthur Cole of the Department of Physics for the use of his electron microscope facilities and for his helpful criticism.     

Electron microscopy of G-banded human mitotic chromosomes

Trypsin-treated human metaphase chromosomes stained with Giemsa and uranyl acetate showed clear, reproducible band structures under the transmission electron microscope (TEM). The banding pattern observed with TEM corresponded very closely to the G-band pattern visualized by light microscopy. The TEM images were used for karyotype analyses. Trypsin-treated chromosomes stained with uranyl acetate alone also showed clear G-bands under TEM. Shadow casting in addition to uranyl acetate staining revealed more structural detail of the chromosomes. Chromosome fibers, 200 Å–300 Å in diameter, were observed in the interband regions. Most chromosomes showed the major G-bands under the higher TEM magnification wit0out any trypsin treatment.     

Fine structure of 33 258 H-treated chromosomes

Summary Metaphase chromosomes of mouse strain L cells show strikingly uncondensed pericentric heterochromatic regions after treatment of living cells with the benzimidazol-derivate 33258 Hoechst. In electron micrographs of total preparations after G-band staining the chromosomes are seen to be made up of irregularly folded fibrils of 200–400 Å in diameter. In the uncondensed regions only very few fibrils laid in loose loops are present, making it probable that only one fibril forms one chromatid.     

The electron-microscopic structure of nucleoprotein fibrils from metaphase chromosomes treated with salt

The diameter of nucleoprotein fibres of metaphase chromosomes is sensitive to salt concentrations. Treatment of human lymphocyte cells in metaphase with a hypotonic medium and spreading them on a water surface causes swelling of the chromosome fibres from 150–180 Å to 230–250 Å. Treatment of the chromosomes with 0.15–0.45 M NaCl, a concentration at which histones are not yet removed from the nucleoprotein complexes, apparently does not affect the chromosome structure. Treatment with NaCl solutions between 0.6 M and 2.0 M NaCl leads to a progressive extraction of the chromosomal proteins and decreases the diameter of the chromosome fibres to 80–100 Å and less.Dedicated to Prof. Dr. A. Butenandt on the occasion of his 70th birthday.     

Elektronenmikroskopische Untersuchungen menschlicher Metaphasen-Chromosomen

Zusammenfassung Chromosomen von Mitosen im Metaphasestadium nach Colchicinbehandlung normaler menschlicher Fibroblastenkulturen wurden mit dem Elektronenmikroskop untersucht.Nach Fixierung in Glutaraldehyd und Einbettung in Epon zeigen Schnittpräparate nach Kontrastierung mit Uranylacetat als feinste erkennbare Elemente etwa 30 Å dicke, schraubig gewundene Fibrillen, die dickere, vielfach und unregelmäßig gefaltete Fibrillen von 100–150 Å Durchmesser aufbauen.Isolierte ganze Chromosomen, die zur Präparation mit hypotoner Salzlösung vorbehandelt, in Alkohol-Essigsäure fixiert und luftgetrocknet wurden, lassen stark gewundene dicke Fibrillen von 200–300 Å durchmesser erkennen, die aus schraubig gewundenen 30 Å dicken Fibrillen bestehen. In Schnittpräparaten von ähnlich vorbehandelten Chromosomen finden sich ebenfalls 200–300 Å dicke Fibrillen, die aus 30 Å dicken feineren Fibrillen in lockerer Anordnung aufgebaut sind. Der größere Durchmesser der dicken Fibrillen in hypoton vorbehandelten Präparaten könnte durch Auflockerung der feinen Fibrillen hervorgerufen sein.In allen Präparaten sind die auch lichtmikroskopisch sichtbaren primären Windungen der Chromatiden angedeutet. Die dickeren Fibrillen lassen sonst keine regelmäßige Anordnung erkennen. Längsunterteilungen im Sinne von Halb- oder Viertelchromatiden sind nicht zu sehen. In Totalpräparaten erscheint die Region des Zentromers weniger dicht, und Kinetochoren sind nicht erkennbar.Es wird die Frage diskutiert, ob nur eine kontinuierliche und vielfach gewundene Fibrille oder mehrere miteinander verflochtene Fibrillen und Stränge ein Chromatid aufbauen.

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Metaphase chromosomes of colchizinized normal human fibroblast cultures were investigated with the electron microscope.Sections of glutaradehyde fixed and epon embedded chromosomes show 30 Å thick coiled fibrils building up folded thicker fibrils of 100–150 Å diameter.Isolated total chromosomes pretreated in hypotonic salt solution, fixed in alcohol-acidic acid and air dried, show also 30 Å thick fibrils coiled into thicker fibrils of 200–300 Å diameter. Sections of similarly treated and epon embedded chromosomes show fibrils of similar dimensions but more loosely coiled than in glutaraldehyde fixed sections.Major coils also seen by light microscopy are noticeable in all preparations. No signs of longitudinal subdivisions of the chromatids are detectable. In whole mount preparations the centromere region appears as less dense and kinetochores cannot be seen.The question is discussed whether one single continuous fibril coiled to a thicker fibril which in turn is irregular folded to a strand laid into the major coils builds up a chromatid, or if many thin fibrils join together to thicker fibrils which again form thicker strands which are finally twisted together to a chromatid.

     

Chromosome fibers studied by a spreading technique

Chromosomes and interphase nuclei can be spread on the surface of water in a simplified Langmuir trough. Interphase nuclei of Triturus erythrocytes display fibers with a diameter of about 250–300 Å. Very similar fibers are seen in metaphase chromosomes of cultured human cells. Fibers from grasshopper spermatocyte chromosomes (prophase) are more variable in diameter, and many fibers thinner than 200 Å extend laterally from the chromosome. In the grasshopper spermatocyte, fibers align in parallel to form plates. It is suggested that the 250–300 Å fibers may represent an inactive state of the chromosome material, and that only the thinner fibers are involved in RNA synthesis. The 250–300 Å fibers may result from the folding or coiling of a thinner fiber having the approximate dimensions of the nucleohistone molecule.     

The basis of chromatin fiber assembly within chromosomes studied by histone-DNA crosslinking followed by trypsin digestion

To determine the structural basis of chromatin assembly that leads to chromosome formation in mitosis, crosslinks were introduced by formaldehyde between contiguous components within chromosomes. Crosslinked stable products were then observed by electronmicroscopy after non-cross-linked portions were briefly digested by trypsin to unfold chromosomes. — When the DNA-histone crosslink was the primary product, trypsin readily unfolded the whole chromosome structure while preserving the 250 Å unit chromatin fiber intact; only a single unit fiber was tracked within the centromere region connecting the arms of each chromatid. When a histone polymer was formed by a prolonged formaldehyde crosslinking, trypsin digestion gave rise to chromatin fibers interacting with others at certain distances, and the typical chromosome structure remained unchanged. Regardless of the degree of crosslinking, there were neither thick supercoiled unit fibers nor proteinaceous cores. — These results suggest that the fiber connection may represent, to some extent, the interacting sites of folded chromatin fibers in situ within chromosomes, and also that the 250 Å unit fibers are the sole, highest structural basis in chromosomes. Since virtually no appreciable histone digestion took place in the crosslinked chromosomes, the observation that even after DNA-histone crosslinking the fiber interacting sites were accessible to trypsin preferentially over other portions, may be consistent with our recent results that the exposed, lysine-rich tails of histones represent such interacting sites.     

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (2⁶–2⁷ chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 Å in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.     

Chromosome segregation and expression of rat immunoglobulins in rat/mouse hybrid myelomas

The chromosome segregation of hybrid myelomas from a fusion of rat immunized spleen cells and the mouse myeloma P3-X63-Ag8 has been analysed. Chromosome loss appears to be nonrandom. Most mouse chromosomes are retained. The rat chromosomes are preferentially lost but a few — particularly 1–5 and 13-are rarely lost. The specific retention of some rat chromosomes explains the stability of rat Ig expression in mouse-rat hybrids. Correlation of chromosome loss and retention and loss of rat heavy and light chains leads us to propose chromosome 14 as coding for the heavy chain.     

The surface and internal structure of metaphase chromatin

Mammalian metaphase chromatin has been isolated by an ultrasonication technique and examined by both surface scanning and high-voltage electron microscopy (H.V.E.M.). By both techniques, rod-like structures 0.5 to 0.8 wide and varying in length from 3 to 5 were seen. Evidence is submitted that these represented parts of metaphase chromosomes. — By scanning microscopy the rods showed repeated patterns of wide and constricted areas. The constrictions were spaced 3,000 to 4,000 Å apart and the entire surface of the rods was covered with smaller rounded projections. In addition, longitudinal grooves were occasionally seen. — H.V.E.M. revealed gross folded and unfolded fibres whose sizes correlated with the surface projections seen by scanning microscopy. In addition, microfibrils, 20–40 Å in diameter were seen. The possibility that these fibrils represent the DNA co-helix is discussed.     

Relationship between relative dry mass and average band width in regions of polytene chromosomes of Drosophila

Whole-mounted polytene chromosomes from Drosophila melanogaster were prepared for high-voltage electron microscopy. Relative dry mass of chromosome regions was estimated by densitometry of electron microscopic negatives. Comparison of dry mass of regions of the male X chromosome with that of regions of associated autosomes established that dry mass values are proportional to DNA content. Relative dry mass values of regions of polytene chromosomes from salivary glands, fat body, and malpighian tubules were correlated with the average diameter of bands in these regions: as mass doubled, band width increased by a factor of approximately 2. To provide a standard for estimating absolute levels of polyteny, band widths were measured for chromosomes representing one major polytene class, 256n. These chromosomes were observed to have an average band width of 0.9 m — These observations provide limits to models of chromatin organization in bands. For each chromatid, this area can accommodate up to five chromatin fibers of 250 Å diameter. This value may represent the extent of folding of a chromatin fiber in an average band. Alternatively, a chromatin fiber of higher-order structure could have a maximum diameter of 560 Å in an average band.     

Bukettförmige Strukturen im Ependym der Regio hypothalamica des III. Ventrikels beim Kaninchen

Zusammenfassung Die Wand des III. Ventrikels (Regio hypothalamica) vom erwachsenen Kaninchen wurde elektronenmikroskopisch untersucht. — Unterschiedliche keulenförmige portsätze, deren Perikaryen in nahen und entfernten Regionen liegen, dringen in den Ventrikel ein. Sie heben eine Ependymzelle ab und bilden eine bukettförmige Struktur. Einige Fortsätze enthalten eine spärliche feingranulierte Masse, andere Bläschen (600–1500 Å) oder Bläschen mit dichtem Kern (450–1200 Å). Inmitten des Buketts kann eine Nervenzelle liegen, die einen dicken Fortsatz gegen das Hypendym schickt. Unterhalb des Buketts findet man eine andere Nervenzelle, die Fortsätze ins Bukett sendet. Zahlreiche in das Bukett eintretende Fortsätze stammen aus entfernteren Kerngebieten. Die funktionelle Bedeutung der Strukturen (Neurosekretion ? Resorption ? Rezeptor ?) wird diskutiert.

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Bouquet-like ependymal structures in the hypothalamic region of the 3rd Ventricle (rabbit)The question of neurosecretion or receptor function

Summary The wall of the III. ventricle (Regio hypothalamica) of the adult rabbit has been investigated electron microscopically. —Various bulblike processes of nerve cells, having their perikarya in close or distant regions, penetrate into the ventricle. They lift an ependymal cell and form a bouquet-like structure. Some of the processes contain a sparse granulated material and others include vesicles (600–1500 Å) or dense-cored vesicles (450–1200 Å). In the middle of the bouquet a perikaryon may be seen extending a thick process towards the hypendyma. Below the bouquet another perikaryon may be found extending several processes into the bouquet. The functional significance of these structures (neurosecretion ? resorption? receptor?) is discussed.

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Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Summary Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.Contribution from the Department of Agronomy and published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2730. This research was supported in part by the USDA/SEA Competitive Research Grant 5901-0410-9-0334-0, USDA/ SEA-CSU Cooperative Research Grant 12-14-5001-265 and Colorado State University Hatch Project. This paper was presented partly at the Fourth International Barley Genetics Symposium, Edinburgh, Scotland, July 22–29, 1981     

Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA.     

Differences in DNA composition along mammalian metaphase chromosomes

Denaturation of chromosomal DNA in situ can be achieved without disruption of chromosomal morphology by heating slides at 25–90° C in 10–95% formamide in SSC. The extent of denaturation is proportional to formamide concentration and temperature. Reassociation of denatured DNA is prevented with formaldehyde. — The DNA in the paracentromeric constrictions in human chromosomes 1, 9 and 16 denatures earlier than in any other regions, as shown by the red colour with acridine orange. When the temperature or formamide concentration is raised a red and green banding pattern emerges in which regions known to stain brightly with quinacrine mustard are red whereas other regions are green. The last regions to turn red are the short arms of some acrocentric chromosomes. Since A+T-rich DNA denatures before G+C-rich DNA, it is inferred that QM-bright areas are rich in A+T. Similar results are obtained with mouse and Microtus agrestis cells. — Reassociation of chromosomal DNA denatured by heat and formamide occurs if no formaldehyde is used. In human cells, kinetic studies on reassociation indicate that the highest degree of repetition is in the DNA of the distal half of the Y chromosome. Next in degree of repetition are the paracentromeric constrictions, the short arm regions of some of the acrocentric chromosomes, and all the centromeric regions. Highly repetitious DNA is found in all mouse centromeric regions except that of the Y chromosome. Constitutively heterochromatic segments of X and Y and the autosomal centromeric regions of Microtus agrestis also contain repetitious DNA. — It is proposed that differential base content and susceptibility to denaturation of DNA contribute to or at least accompany Q-, G- and R-banding. The degree of C-banding is related to repetitious DNA. The human Y chromosomal DNA is probably A+T-rich and exceptionally repetitious, exhibiting spontaneous reassociation under many experimental conditions.     

Functional organization of the nucleus in the oogenesis of Chrysopa perla L. (Insecta,Neuroptera)

On the basis of light, autoradiographic (uridine-³H incorporation) and electron microscopic investigation changes of nuclear structures were examined during the oogenesis in Chrysopa perla L. — In early meiotic prophase the oocyte nuclei were found to contain a large body of extrachromosomal DNA. In certain cases the latter splits up into several DNA clumps giving rise to a few (4–7) primary nucleoli, 3–5 in diameter. The primary nucleoli consist of densely packed fibrils 50–100 Å thick. They contain no granular component and are inactive in RNA synthesis. — At the beginning of large growth the extrachromosomal DNA bodies disappear and numerous electron-dense clumps, 0,5–1 in diameter, appear in the nucleus. Instead of the primary nucleoli, the nucleus now contains a great number of ring nucleoli about 0,5–1 in diameter with a granular component (granules are 150 Å). The space between them is filled up with nucleolar strands running from the surface of the ring nucleoli. — At the stage ring nucleoli of uridine^–3 H incorporation into the oocyte nucleus begins. — During later previtellogenesis and at the beginning of vitellogenesis the ring nucleoli disappear and the nucleus is filled with the network of nucleolar strands. Among them there are specific complexes. These consist of electron dense masses, of granular clusters (granules 500 Å in diameter) and large fibrillar electron light bodies. At this stage the nucleus takes the most active part in RNA synthesis. — The process of karyosphere capsule formation was studied by electron microscopy. The capsule was found to be of fibrillar nature; its structure is very peculiar and unlike any known membrane components of the cell. On the basis of cytochemical evidences the characteristics of the capsule are given. — The development of a powerful nucleolar apparatus based on the extrachromosomal DNA and a possible role of the synaptonemal complex and extrachromosomal DNA in formation of the karyosphere capsule is discussed.     

Organization of human mitotic chromosomes

Summary Examinations of human mitotic chromosomes using an electron microscope since the last review in Humangenetik (Schwarzacher, 1970) were summarized. Three methods were used for preparation: ultrathinnsectioning, spreading- and critical point drying and a method for comparing cells in the light and electron microscope.These three methods showed that fibrils are the main elements of organization of chromosomes. Fibrils with a diameter of 20–40 Å, of 100 Å, of 250 Å and thick fibrils (bundles) of 500–1000 Å thickness were described.A comparison of chromosomes in the light and electron microscope showed, that metaphase chromosomes can be characterized by the number of their primary coils.Examinations of Giemsa-banding techniques with electron microscope showed fibrils as being clearly visible. G bands are coils of thick fibrils (up to 1000 Å).The methods based on these new results were discussed.

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Zusammenfassung Es wurde der Stand der Untersuchung menschlicher Mitosechromosomen im Elektronenmikroskop seit der letzten in Humangenetik erschienenen zusammenfassenden Arbeit (Schwarzacher, 1970) behandelt. Drei Methoden wurden bei der Präparation angewandt: Ultradünnschnittechnik, Spreitungs- und Kritischer-Punkt-Trocknungstechnik und vergleichende licht- und elektronenmikroskopische Methode.Alle drei Methoden zeigten, daß Fibrillen wesentlich am Bau von Chromosomen beteiligt sind. Es wurden Fibrillen mit einem Durchmesser zwischen 20 und 40 Å, Fibrillen mit ca. 100 Å, Fibrillen mit 250 Å und dicke Fibrillen (Bündel) mit 500–1000 Å Durchmesser beschrieben.Vergleichende licht- und elektronenmikroskopische Techniken zeigten, daß Metaphasechromosomen durch ihre Primärwindungen zu charakterisieren sind.Untersuchungen der Giemsabandentechniken im Elektronenmikroskop ergaben, daß Fibrillen deutlicher zur Darstellung kommen. G-Banden imponieren als Coils aus dicken Fibrillen (bis 1000 Å) aufgebaut.Aus den neuen Befunden resultierende Modelle werden diskutiert.

     

DNA replication sequence of human chromosomes in blood cultures

Summary The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G ₂ periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H³. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S₁ to S₄.The first chromosomes to replicate were Nos. 1, 3, 5 and X followed by the Nos. 2, 4 and several chromosomes of groups 6–12, 13–15 and 19–20. Later the pairs 16, 17, 18 and the chromosomes of group 21–22 replicated. Chromosome Y in the male was the last to replicate, beginning its duplication when all the other chromosomes had reached the intermediate S stage.The earliest chromosomes to finish the duplication were Nos. 19, 20 and 21 followed by Nos. 16, 17, 18, 22 and the chromosomes of group 13–15. Afterward and at about the same time the replication of pairs 2, 4, 6, 8, the X and Y chromosomes in the male and one X chromosome in the female concluded. The other X chromosome in the female was the last to end its duplication appearing totally labelled until the final stage of the S period.Replication of the long and medium size chromosomes begins at localised regions, then extends over the total length of the chromosome and at the end of the S stage takes place only in small zones different from those replicating early.Asynchrony between homologous chromosomes was observed at the beginning and at the end of the S period.     

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target—recognizing that we would not produce a single chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1:29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome (Chr) 1q12–q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43–q46, near the terminal end.