Nonrandom chromosome arrangements in germ line nuclei of Sciara coprophila males: the basis for nonrandom chromosome segregation on the meiosis I spindle.

Meiosis I in males of the Dipteran Sciara coprophila results in the nonrandom distribution of maternally and paternally derived chromosome sets to the two division products. Based on an earlier study (Kubai, D.F. 1982. J. Cell Biol. 93:655-669), I suggested that the meiosis I spindle does not play a direct role in the nonrandom sorting of chromosomes but that, instead, haploid sets are already separated in prophase nuclei well before the onset of spindle formation. Here I report more direct evidence that this hypothesis is true; this evidence was gained from ultrastructural reconstruction analyses of the arrangement of chromosomes in germ line nuclei (prophase nuclei in spermatogonia and spermatocytes) of males heterozygous for an X-autosome chromosome translocation. Because of this translocation, the maternal and paternal chromosome sets are distinguishable, so it is possible to demonstrate that (a) the two haploid chromosome sets occupy distinct maternal and paternal nuclear compartments and that (b) nuclei are oriented so that the two haploid chromosome sets have consistent relationships to a well-defined cellular axis. The consequences of such nonrandom aspects of nuclear structure for chromosome behavior on premeiotic and meiotic spindles are discussed.

Meiosis in Sciara coprophila: structure of the spindle and chromosome behavior during the first meiotic division.

Light microscope descriptions of meiosis I in males of the fungus gnat Sciara coprophila suggested the presence of a monopolar spindle in which maternal and limited chromosomes move poleward while paternal chromosomes "back away" from the pole. The ultrastructural analysis reported here, based upon serial sections of cells in different stages of meiosis I, shows that the spindle is indeed monopolar with a distinctive differentiation, the polar complex, at one pole. This complex is the focus of a conical radiation of spindle microtubules. Kinetochores of paternal chromosomes face the complex and microtubules associated with these kinetochores run toward the complex. No kinetochore microtubules were discovered on maternal or limited chromosomes. When the position of paternal, maternal, and limited chromosomes is compared at various stages, it is found that limited chromosomes always remain near the polar complex, paternal chromosomes remain far from it and only maternal chromosomes move closer to the pole. Apparently, chromosome segregation does not depend on paternal chromosomes "backing away" from the pole, and the required movement of maternal chromosomes take place in the absence of kinetochore microtubules. In the prophase nucleus, limited and maternal chromosomes are already spatially separate from paternal chromosomes before the spindle forms. Thus, the monopolar spindle functions only to increase the distance between already segregated sets of chromosomes. An extensive system of microtubule-associated membranes outlines the spindle; the possibility that maternal chromosome movement is somehow related to the presence of this membrane is discussed.

An unusual mitotic mechanism in the parasitic protozoan Syndinium sp.

Syndinium and related organisms which parasitize a number of invertebrates have been classified with dinoflagellates on the basis of the morphology of their zoospores. We demonstrate here that with respect to chromosome structure and chemistry as well as nuclear division, they differ fundamentally from free-living dinoflagellates. Alkaline fast green staining indicates the presence of basic proteins in Syndinium chromosomes. Chromatin fibers are about 30 A thick and do not show the arrangement characteristic of dinoflagellate chromosomes. The four V-shaped chromosomes are permanently attached at their apexes to a specific area of the nuclear membrane through a kinetochore-like trilaminar disk inserted into an opening of the membrane. Microtubules connect the outer dense layer of each kinetochore to the bases of the two centrioles located in a pocket-shaped invagination of the nuclear envelope. During division kinetochores duplicate, and each sister kinetochore becomes attached to a different centriole. As the centrioles move apart, apparently pushed by a bundle of elongating microtubules (central spindle), the daughter chromosomes are passively pulled apart. During the process of elongation of the central spindle, the cytoplasmic groove on the nuclear surface which contains the central spindle sinks into the nuclear space and is transformed into a cylindrical cytoplasmic channel. A constriction in the persisting nuclear envelope leads to the formation of two daughter nuclei.

Division in the dinoflagellate Gyrodinium cohnii (Schiller). A new type of nuclear reproduction.

Dinoflagellates are of interest because their chromosomes resemble the nucleoplasm of prokaryotes both chemically and ultrastructurally. We have studied nuclear division in the dinoflagellate Gyrodinium cohnii (Schiller), using cells obtained from cultures undergoing phasic growth. Electron micrographs of serial sections were used to prepare three-dimensional reconstructions of nuclei and chromosomes at various stages of nuclear division. During division, a complex process of invagination of the intact nuclear envelope takes place at one side of the nucleus and results in the formation of parallel cylindrical cytoplasmic channels through the nucleus. These invaginations contain bundles of microtubules, and each of the bundles comes to lie in the cytoplasm of a cylindrical channel. Nuclear constriction occurs perpendicular to these channels without displacement of the microtubules. There are no associations between chromosomes and the cytoplasmic microtubules. In dividing cells most chromosomes become V-shaped, and the apices of the V's make contact with the membrane surrounding cytoplasmic channels. It is proposed that the membrane surrounding cytoplasmic channels in the dividing nucleus may be involved in the separation of daughter chromosomes. Thus, dinoflagellates may resemble prokaryotes in the manner of genophore separation as well as in genophore chemistry and ultrastructure.

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study.

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.

Spindle microtubules and their mechanical associations after micromanipulation in anaphase.

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.

Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation.

The relationship between chromosome movement and microtubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.

Isolation and characterization of phi80dgal transducing phages that carry gal operator-promoter insertion mutations.

phi80dgal transducing bacteriophages have been isolated by the F-fusion technique of Press et al. (1971) and gal-operator-promoter insertion mutations have been introduced by homogenate formation. Five different phi80dgal isolates have been studied in more detail. One of the phi80 phages transduces the gal operon and gene aroG as well as at least part of the trp-operon; the gal operon of another phi80dgal transducing phage is inverted with respect to the phi80dgal sequences. Heteroduplex DNA mapping indicates that one of the phi80dgal isolates in addition to the gal operon and a portion of the adjacent chromosomal region carries an IS2-element which is derived from the F'gal episome. The isolated phi80dgal phages may be utilized for preparing pure gal mRNA and insertion-RNA as well as pure gal operon DNA.

Electron microscopy of the spindle in locally heated cells.

Individual living cells in metaphase were exposed to a steep temperature gradient by placing a microheater near one spindle pole. The cells were then fixed and the spindle was examined by electron microscopy. The structure of the warmer half-spindle differed from the cooler half-spindle in several ways. Kinetochore microtubules were nearly parallel in the warmer half-spindle but were divergent in the cooler. The total length of microtubules in the warmer half-spindle was 52 per cent greater and the number of kinetochore microtubules per kinetochore averaged 16 per cent higher than in the cooler half-spindle. The warmer half-spindle was longer than the cooler. These observations clearly demonstrate a locally enhanced assembly of microtubules in the warmer half-spindle. The electron microscope study makes still clearer the unusual character of chromosome movement in the differentially heated cells: the structure of the warmer half-spindle is hard to distinguish from that in normal cells, yet chromosome movement there is far slower than normal (Nicklas, 1979).

IS1 and IS2 mutations in the ribosomal protein genes of E. coli K-12.

The insertion mutations I15 and I16 in the ribosomal protein gene cluster at 64 min in Escherichia coli are identified as IS1 and IS2 using electron microscope heteroduplex analysis.

Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes.

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.