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At least 19 recordsLinked to original sources
PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗
PubMed1992

Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malignant melanoma.

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.

Base Sequence↗
PubMed1979

Micropreparation techniques in quantitative histochemistry - density gradient centrifugation, manual microdissection and laser microbeam preparation of tissue.

Each quantitative histochemical problem needs its specific method for tissue preparation. In this connection two of the most important preparation methods, density gradient centrifugation and microdissection of freeze-dried tissue slices, are described. Density gradient centrifugation is a very effective procedure for preparative separation of cell particles such as cell nuclei. The details of the preparation of glial and neuronal cell nuclei are described. The in vitro phosphorylation of histone in the chromatin in relation to age is given as a practical example of the quantitative histochemical application to a preparation of cell nuclei. Other techniques of tissue preparation are the manual tissue microdissection according to Lowry and the Laser microbeam preparation. Advantages and disadvantages of both methods are compared. It is shown, that the introduction of Laser microbeam dissection technique, as alternative to manual microdissection, add new dimensions to Lowry's ultramicrochemical methods. One has greater freedom in the choice of the sample size and the number of samples dissected from the same slice. Furthermore, the need for a well-trained person for the preparation is eliminated. The preparation is also considerably less time consuming and easier to perform than the manual free hand preparation. Two quantitative histochemical methods used for the investigation of microdissected tissue samples are described: the gas-chromatography-massfragmentography (GC/MS)-method for determination of transmitters and its metabolites as well as the enzymatic cycling technique of Lowry. The GC/MS-method is explained with an example of noradrenaline and dopamine determination. The enzymatic cycling technique is demonstrated in combination with the Oil-Well-Technique for determination of the NADP-cycle.

Cell Fractionation↗
PubMed1987

A new microdissection technique for regional biochemical analysis of the rat spinal cord: serotonin, norepinephrine, dopamine and uric acid.

This report described a new microdissection procedure to evaluate the regional distribution of neuromediators in the rat spinal cord. Different segments are first divided into sagittal slices. From these, different grey and white matter regions can be microdissected. This permits selective biochemical measurements in different laminae, including the area around the central canal. White and grey matter can also be differentially analysed. Using HPLC with electrochemical detection, we report on the regional analysis of biogenic amines as well as uric acid. An increase in 5-hydroxytryptamine (5-HT) levels was observed from cervical to lumbar segments. 5-HT levels were highest in the motoneurone samples (lamina IX) of the lumbar cord. The next highest levels were found in lamina X, followed by the intermediate grey matter laminae and the dorsal horn. Small amounts of 5-HT were detected in the white matter. Interestingly, the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT was greatest in the white matter and least in the motoneurone sample. Norepinephrine (NE) levels were higher in the thoracic than in the cervical or the lumbar cord. The highest levels were found in the lamina X in thoracic segments. With this exception, no marked laminar difference in NE levels was observed. Dopamine (DA) levels were highest in the dorsal horn of the cervical and the thoracic cord, the next highest levels were found in the intermediate grey matter and lamina X in the same segments. The distribution of uric acid (UA) was comparable to that of NE: UA levels were highest in the thoracic cord, but no marked laminar difference was observed. On the other hand, UA levels in white matter generally exceeded those in the grey matter. These data indicate the value of a sagittal regional microdissection of the spinal cord. The ability to separately analyse different laminae of the cord (as well as differentiating grey and white matter) should prove useful in future studies of experimentally evoked changes in neurotransmitters within functionally distinct regions of the spinal cord.

Animals↗
PubMed1991

Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome.

We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.

Base Sequence↗
PubMed1985

ATP and cAMP system in the in vitro response of microdissected cortical tubules to PTH.

Responsiveness of proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) microdissected from mouse kidney to PTH, in terms of cAMP accumulation and stimulation of adenylate cyclase, was examined. In both PCT and DCT, the cell-free adenylate cyclase was stimulated at least 10-fold by the same dose (10 U/ml) of PTH, and activity of cAMP phosphodiesterase was about 80% higher in DCT than in PCT. In intact tubules, while the incubation with PTH increased cAMP content in DCT more than 10-fold, it failed to increase the cAMP levels in PCT. To explain discrepancies between cell-free and intact cell incubations, ATP content in microdissected tubules was determined with use of a microbioluminescence assay. ATP content in PCT (4.0 +/- 1.3 fmol/mm, n = 30) was dramatically lower than ATP content of DCT (376.8 +/- 54.3 fmol/mm, n = 25). Incubation with 1 microM rotenone reduced markedly (delta -98%) the ATP content in DCT. In DCT, with ATP depleted by 1 microM rotenone, PTH failed to increase the cAMP, although 1 microM rotenone did not inhibit the adenylate cyclase activity. When 0.1 mM of 1-methyl-3-isobutylxanthine (MIX) was added to the incubation medium, PTH caused a marked elevation in tubular cAMP in PCT and to even a greater degree in DCT. Present results show that various segments of microdissected tubules differ greatly in their ability to maintain adequate ATP levels for cAMP generation in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Effects of atrial natriuretic factor on cyclic guanosine monophosphate and cyclic adenosine monophosphate accumulation in microdissected nephron segments from rats.

Atrial natriuretic factor (ANF) (1 microM) markedly increased cyclic guanosine monophosphate (cGMP) content in microdissected glomeruli (35-fold) and in microdissected inner medullary collecting ducts (IMCD) (20-fold). ANF caused little or no increase in cGMP content in other nephron segments. The threshold concentration for increased cGMP accumulation by ANF was 0.1-1 nM in IMCD, which is in the range reported for rat plasma. Sodium nitroprusside (1 mM), which selectively stimulates soluble guanylate cyclase, increased cGMP content in glomeruli but not in IMCD. ANF did not alter cAMP accumulation in the absence or presence of vasopressin (AVP) or parathyroid hormone (PTH) in outer and inner medullary tubule suspensions, or in microdissected proximal convoluted tubules (PCT), medullary thick ascending limbs (MAL) or IMCD. These data are compatible with the hypothesis that cGMP is a second messenger for a physiologic action of ANF in the inner medullary collecting duct. ANF apparently activates membrane-bound guanylate cyclase in this segment.

Animals↗
PubMedDate not supplied

Use of microdissected airways to define metabolism and cytotoxicity in murine bronchiolar epithelium.

The use of the mouse for carcinogenesis bioassays has raised questions regarding the cell of origin of lung tumors. Since a feature of chronic lung injury from aromatic hydrocarbons is an apparent alteration in target cell susceptibility, the present study was designed to test the feasibility of using microdissected pulmonary airways to evaluate the metabolism and cytotoxic response of one of the potential targets of pulmonary carcinogens, the bronchiolar Clara cell. Airways were microdissected from mouse lungs that had been filled by injection of agarose (1%) into the trachea. Ultrastructural integrity of the explants has been maintained for up to 8 h in culture. The cytotoxic response of bronchiolar epithelium in explants incubated with naphthalene (0.5 mM) was identical to the vacuolation and exfoliation observed in bronchioles of mice 24 h after intraperitoneal administration of naphthalene (100 or 300 mg/kg). Pre-incubation of the explants with piperonyl butoxide, a cytochrome P-450 monooxygenase inhibitor, prevented naphthalene-induced cytotoxicity. Naphthalene monooxygenase activity was easily measurable in all levels of airway, including trachea, lobar bronchi, major and minor daughter pathways, and distal bronchioles. No metabolism was detected in lung parenchyma or large vessels. Dihydrodiol and a glutathione adduct derived from 1R, 2S-naphthalene oxide were the sole metabolites detected by HPLC in incubations of airway explants. Formation of a single diastereomeric glutathione conjugate indicated that the metabolic epoxidation of naphthalene was highly stereoselective. Glutathione S-transferase activity was measured in all compartments, with the highest activities in trachea and lowest in distal bronchiole and pulmonary vein. Explants maintained pools of reduced glutathione for up to 4 h in culture. We conclude that microdissected airways have excellent potential for: (1) defining the capability of bronchiolar epithelium to catalyze xenobiotic biotransformation, (2) comparing activity in target and nontarget lung compartments as a means of identifying specific metabolic pathways associated with the cytotoxic response, and (3) use with a variety of species, including nonhuman primates and humans, as a means of providing appropriate data for extrapolation of effects in the intact animal to the human, where bioassay is not possible.

Animals↗
PubMed1990

Microdissection of the fragile X region.

We have microdissected and cloned the region around the fragile site at Xq27.3 on the human X chromosome. All of the clones tested map to the Xq27-Xq28 region, and detailed mapping on a panel of somatic cell hybrids indicates that the microdissected library contains sequences derived from both sides of the fragile X mutation. Some of these clones give signals in rodent DNA. This library demonstrates the power of microdissection for the identification of potential coding sequences near a disease locus and provides a promising resource for the identification of the fragile X mutation.

Animals↗
PubMed1991

Detection of APC region-specific signals by nonisotopic chromosomal in situ suppression (CISS)-hybridization using a microdissection library as a probe.

The chromosome region 5q22 harbouring the putative gene associated with adenomatous polyposis coli (APC) was microdissected and microcloned from GTG-banded human metaphase chromosomes. In order to determine the precise regional localization of the microdissected material, we used polymerase chain reaction amplified microclones as a bulk-probe in nonradioactive chromosomal in situ suppression hybridization of human metaphase spreads. Specific in situ hybridization signals were obtained on the long arm of chromosome 5 in accordance with the chromosomal region excised for the cloning procedure. The application of this detection system should provide a rapid and powerful tool for analyzing patients with translocations or microdeletions of a given chromosome region.

Adenomatous Polyposis Coli↗
PubMed1990

Microdissection of banded human chromosomes.

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 1250X) 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.

Cells, Cultured↗
PubMed1976

The laser in the Lowry technique for microdissection of freeze-dried tissue slices.

A new technique for tissue microdissection is described. This procedure, using an u.v.-laser micropreparation instrument, overcomes the extremely time-consuming manual preparation. The u.v.-laser micropreparation design allows fast, precise, reproducible and smear-contamination-free tissue microdissection. The preparation of the tissue sample can be programmed by tracing out the area to be sampled with a non-destructive 0.5 mW He-Ne-laser aiming-beam. The tract is stored in a small electronic unit, which then guides the motor-driven stepping stage on the microscope in the actual dissection run with the u.v.-laser. The laser power is adjustable in the range 4 to 40 kW and controlled by a photo diode displayed on an oscilloscope screen. In the tissue slice, prepared according to Lowry, an unlimited number of cells or tissue compartments can be dissected and afterwards weighed. The procedure described offers a broader use of the quantitative microhistochemical techniques of Lowry and of Neuhoff.

Acid Phosphatase↗
PubMed1990

Microdissection and microcloning of human chromosome 7q22-32 region.

Genetic information contributing to cystic fibrosis in addition to the CF gene is suggested to reside on the long arm of the human chromosome 7. In our attempt to analyze this genomic region in detail, we generated a region-specific DNA probe library by microdissection and microcloning of the midpiece of the chromosome 7q arm. Microdissection was performed in unstained metaphase spreads from a human x mouse hybrid cell line containing chromosome 7 as the only human chromosome. We obtained 593 clones from 75 dissected chromosomal fragments. At least 88% of the microclones were true recombinants; 40% of the clones contained repetitive sequences as determined by plaque hybridization with genomic DNA as probe. The overall mean fragment size of insert fragments was 3.2 kb, the median size was 3.5 kb. Regional mapping of 30 DNA fragments was performed by the aid of hybrid cell lines containing different segments of human chromosome 7; 50% of the microcloned inserts were found to map to 7q22-32.

Animals↗
PubMed1986

Microdissection studies on the polarity of unequal division in grasshopper neuroblasts. I. Subsequent divisions in neuroblast-type cells produced against the polarity by micromanipulation.

Equal or unequal division against the polarity of normal division was induced in grasshopper neuroblasts by means of a microdissection technique. The subsequent cell divisions were traced in order to analyse the factors that determine the polarity. Daughter cells of two types (neuroblast-type and ganglion cell type) were produced by operations in which the mitotic apparatus was rotated or shifted. Cell types were classified by such characteristics as nuclear shape, mitotic activity, and inequality or equality of the subsequent cytokinesis. It became evident that the fate of daughter cells was determined simply by the cytoplasmic volume. In 27 cases out of 40 microdissecting operations, both sister cells were recognized as of neuroblast type. Mitosis of these neuroblast-type sister cells proceeded asynchronously. The time required for neuroblast-type cells to reach metaphase of the second division depended on their volume. It is considered that the polarity of unequal division in grasshopper neuroblasts may be maintained by a joint action of the cap cells attaching to one of the polar regions of the cell and the cortex differentiated in the previous cell division.

Animals↗
PubMed1976

Microdissection of peripheral nerve: collagen and lipid distribution with morphological correlation.

Peripheral nerve is a complex tissue composed of endoneurial fascicles surrounded by perineurium and epineurium. We separated endoneurium from peri- and epineurium in human sural nerves by "endoneurial plucking", a method of microdissection. Endoneurial contents (axons, myelin sheaths, Schwann cells, vessels, and interstitial collagen) were cleanly separated in high yield from enveloping connective tissue, by both microscopic and biochemical criteria. Most of the nerve sulfatide and unesterified sterol was found in the endoneurial fraction while most of the collagen was in the peri-epineurial fraction. This microdissection method should prove useful in biochemical investigations of peripheral nerve.

Adult↗
PubMed1991

Laser microdissection of the fragile X region: identification of cosmid clones and of conserved sequences in this region.

Laser microdissection has been used to dissect material from the X-chromosome region involved in fragile-X-linked mental retardation. After dissection, single chromosome slices corresponding to this fragile site were subjected to DNA amplification using either a vector ligation method (to provide known anchor sequences) or primer oligonucleotides corresponding to the ubiquitous Alu sequences. Amplified material was then cloned or, alternately, used to screen a gridded cosmid library. Eight cosmid clones identified in this way were regionally mapped using a panel of hybrid cell lines and shown to originate from a narrow interval centered on the fragile X site. Two clones are included in the approximately 6-cM interval defined by probes RNI (DXS369, 5 cM proximal) and VK21 (DXS 296, 1-2 cM distal) and which includes the fragile site, and at least one clone contains sequences conserved across species suggestive of a gene. This method combines the focused approach of microdissection and the convenience of obtaining cosmid (rather than small-insert) clones; it may be useful for studies of other defined chromosomal regions.

Animals↗
PubMed1991

New markers for the neurofibromatosis-2 region generated by microdissection of chromosome 22.

To identify new DNA markers around the neurofibromatosis-2 gene on human chromosome 22, the critical region (22q12-q13.1) was microdissected and microcloned from GTG-banded metaphase chromosomes. Eighteen thousand recombinant clones were obtained. Twenty-seven of 55 clones tested (50%) detected single-copy DNA sequences. Nine of nine clones analyzed in detail were found to map to chromosome 22. Interestingly one clone (EAN04) is part of the leukemia inhibitory factor gene which has previously been mapped to 22q11.2-q13.1. Four clones (EAN01, EAN47, EAN57, and EAN68) detect DNA polymorphisms. These probes were used to compare constitutional and tumor genotypes of 41 patients with acoustic neurinoma. Loss of constitutional heterozygosity was identified in 17 of 31 informative cases (55%). From our data we conclude that the microdissection library is a valuable resource for physical and genetic mapping studies in neurofibromatosis-2.

Chromosome Mapping↗
PubMed1992

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗