Microdissection of plant chromosomes by argon-ion laser beam

Summary Rice and barley chromosomal samples were prepared both on a polyester membrane and on an ordinary glass slide and subjected to microdissection by an argon-ion laser. The intensity and the position of the laser beam were controlled by a microcomputer. The most suitable intensity to obtain chromosomal fragments was determined experimentally. As a result, specific regions of the centromere, satellite, short arm, or long arm, of the barley and rice chromosomes were dissected out from the chromosomal spreads. Chromosomal fragments were also successfully transferred from the sample into an Eppendorf tube.     

Microdissection of banded human chromosomes

Summary Physical dissection of metaphase chromosomes is the most straightforward approach for the isolation of DNA sequences from specific chromosome regions. However, conventional microdissection techniques are too crude and inefficient for analysis of the human genome. Here we describe a technique for the precise dissection of single bands from GTG-banded chromosomes. Cells from normal amniotic fluid cell cultures are harvested by the pipette method. Microdissection is performed on an inverted microscope (magnification 1250 x) with the help of extended siliconized glass needles and an electronically controlled micromanipulator. Enzymatic amplification of the dissected DNA allows the construction of band-specific DNA libraries from as few as 20 dissected chromosome fragments.     

Microdissection and microcloning of plant chromosomes

Cytogenetic and molecular tools play an increasingly important role in plant genome research. A number of interesting applications that involve chromosome painting, the relationship between specific chromosomes and specific linkage groups, the relationships between physical and genetic distances on linkage maps, and the isolation of genes of interest, have been the subjects of recently published research. The aim of this paper is to review the different techniques available for chromosome microdissection and microcloning, and their use for the study of plant genomes. The quality of chromosomal DNA obtained is considered, and some recent results from our laboratory are presented.     

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.     

Genetic changes induced in higher plant cells by a laser microbeam

Introducing foreign genes into higher plants has proved to be complicated, with the exception of transformation of protoplasts of some plants (Negrutiu et al. 1987). In particular, culture of protoplasts and regeneration to plants are difficult in many monocotyledonous crops. Therefore, it would be desirable to avoid extensive tissue culture by introducing cloned genes directly into cells. A laser microbeam can perforate plant cell walls, thus facilitating uptake of genes into cells.     

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.     

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a λ vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes. This paper is dedicated to Professor W. Beermann on the occasion of his sixtieth birthday     

A laser microbeam study of amoeboid movement

Specimens of Amoeba proteus and Chaos carolinensis were vitally stained with Alcian blue and subjected to pulsed laser microbeam irradiation in the flux density range of 0.5–4.0 joules/cm². Sublytic doses of microbeam irradiation influenced streaming and cell movement only in vitally stained cells and when delivered to the frontal regions of pseudopodia. The effect was temporary, as streaming resumed after some seconds. Exposure of the uroid to similar amounts of radiation undoubtedly had a stronger heating effect because of greater dye accumulation in the uroid; however, there was no detectable instantaneous or delayed effect of irradiation of the uroid. Higher doses of irradiation in Marshall's medium cause rupture of the plasmalemma and rapid flow of cytoplasm from the point of injury, irrespective of its location, followed in 1.0–1.2 sec by an elastic recoil. The initial rapid flow toward the wound was apparently caused by a slightly higher hydrostatic pressure within the cell because it was reduced in 0.25 M sucrose. When the uroid was suddenly opened to the sucrose medium by a lytic dose of microbeam irradiation, cytoplasm continued to flow into extending pseudopodia. Present results are not only consistent with the frontal contraction theory, but indicate that it is unnecessary to postulate an additional contraction of the uroid as a contributing factor in steady-state amoeboid movement. The results in addition shed some light on the speed of the surface precipitation reaction in wound-healing, on the existence of an internal hydrostatic pressure but not a pressure gradient along the axis of movement and on the viscoelastic properties of the cytoplasm.     

Laser microbeam irradiation of the juxtanucleolar region of prophase nucleolar chromosomes

To further understand the function of the nucleolus organizer (NO), especially as it relates to the mitotic cycle, we extended our previous irradiation studies to prophase chromosomes and nucleoli. The juxtanucleolar region of nucleolar chromosomes was irradiated with the argon laser microbeam, and cells were observed for several days. Nuclei with two nucleoli were generally chosen for irradiation because of their two clear secondary constrictions. Summarized results are as follows: (1) When either one or several juxtanucleolar sites of both or all nucleoli are irradiated, the mitotic process is blocked and the cells return to interphase. (2) When only the chromosomes associated with the largest nucleolus are irradiated, mitosis is also blocked. (3) When the juxtanucleolar regions of the smallest nucleolus are irradiated, the cells generally go into metaphase and complete division, but with a reduction in the number of resulting nucleoli. (4) When the nucleoli themselves are irradiated, mitosis proceeds and daughter nuclei show no reduction in nucleolar number. (5) When chromosomes are randomly irradiated at non-juxtanucleolar regions, the nucleus divides and produces the same number of nucleoli in each daughter nucleus as were present in the mother cell.     

The movement of melanosomes in melanophore fragments obtained by laser microbeam irradiation

The melanophores of the teleost Gymnocorymbus ternetzi are filled with pigment granules, melanosomes, which in response to appropriate treatments, can disperse throughout the cytoplasm or form an aggregate in the cell center. Melanophores with the dispersed pigment were irradiated by a laser microbeam, focused on the cell center by the microscope objective. If the average energy of the microbeam was 6-7 microJ, either the center of the melanophore was damaged and a single ring-shaped fragment was formed, or the cell was broken into several fragments of smaller size. The fragments retained their ability to move the pigment granules. In ring-shaped fragments, after adrenaline treatment, the melanosomes formed a ring-shaped aggregate moving away from both outer and inner (irradiation-produced) margins of the fragment. The smaller fragments treated with adrenaline moved the pigment to their centers. Both small and ring-shaped fragments could aggregate melanosomes as soon as 5 minutes after irradiation.     

Structural analysis of human hair single fibres by scanning microbeam SAXS

The origin of the curliness of human hair was revealed by scanning microbeam small angle X-ray scattering (SAXS), based on the nanostructure of keratin fibre arrangement. Scanning microbeam SAXS patterns of single hair fibres have been measured across the fibres and the differences in the patterns between the inner and the outer sides of the curvature were successfully detected. The analysis of the equatorial and azimuthal scattering intensity profiles showed that the arrangement of the intermediate filaments was different between the inner and the outer sides of the curvature. From the analogy with Merino and Romny wool, it is suggested that different types of cortices exist in human hair. It is concluded that, regardless of the ethnic origins (African, Caucasian, and Asian), the macroscopic curl shape of the hair fibre originate from the inhomogeneity of the internal nanostructure, arising from inhomogeneous distribution of two types of cortices.