scholarly journals Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

1988 ◽  
Vol 80 (3) ◽  
pp. 224-234 ◽  
Author(s):  
P. Lichter ◽  
T. Cremer ◽  
J. Borden ◽  
L. Manuelidis ◽  
D. C. Ward
Keyword(s):  
Recombinant Dna ◽  
Human Chromosomes ◽  
Dna Libraries ◽  
Interphase Cells ◽  
Individual Human

Progress in the molecular cytogenetics of man

1988 ◽  
Vol 319 (1194) ◽  
pp. 239-248 ◽  
Keyword(s):  
Dna Sequences ◽  
Recombinant Dna ◽  
Molecular Cytogenetics ◽  
Chromosome Analysis ◽  
Parental Origin ◽  
Human Chromosomes ◽  
Recombinant Dna Technology ◽  
Chromosome Deletions ◽  
Dna Technology

Recombinant DNA technology has contributed greatly to the precision of chromosome analysis in man. Breakpoints of chromosome deletions and rearrangements may be defined on a chromosome map whose landmarks are the loci of DNA sequences rather than Giemsa bands. Flow cytogenetics allows the extent of chromosome duplications and deletions to be measured more precisely than has hitherto been possible. DNA probes can reveal hidden translocations through the application of in situ hybridization, and may be used as markers to determine the parental origin of non-disjunction. It is evident that a study of the pathology of human chromosomes now requires the combined skills of recombinant DNA and cytology.

scholarly journals Selective transfer of individual human chromosomes to recipient cells.

1985 ◽  
Vol 5 (1) ◽  
pp. 140-146 ◽  
Author(s):  
P J Saxon ◽  
E S Srivatsan ◽  
G V Leipzig ◽  
J H Sameshima ◽  
E J Stanbridge
Keyword(s):  
Hypoxanthine Phosphoribosyl Transferase ◽  
Human Chromosomes ◽  
Isoenzyme Analysis ◽  
Phosphoribosyl Transferase ◽  
Microcell Hybrid ◽  
Multiple Copies ◽  
G 11 ◽  
Hybrid Clones ◽  
Individual Human

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.

Selective transfer of individual human chromosomes to recipient cells

1985 ◽  
Vol 5 (1) ◽  
pp. 140-146
Author(s):  
P J Saxon ◽  
E S Srivatsan ◽  
G V Leipzig ◽  
J H Sameshima ◽  
E J Stanbridge
Keyword(s):  
Hypoxanthine Phosphoribosyl Transferase ◽  
Human Chromosomes ◽  
Isoenzyme Analysis ◽  
Phosphoribosyl Transferase ◽  
Microcell Hybrid ◽  
Multiple Copies ◽  
G 11 ◽  
Hybrid Clones ◽  
Individual Human

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

scholarly journals Simultaneous genotypic and immunophenotypic analysis of interphase cells using dual-color fluorescence: a demonstration of lineage involvement in polycythemia vera

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 1033-1038 ◽  
Author(s):  
CM Price ◽  
EJ Kanfer ◽  
SM Colman ◽  
N Westwood ◽  
AJ Barrett ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Abnormalities ◽  
Myeloproliferative Disorders ◽  
Chromosome 8 ◽  
Dual Fluorescence ◽  
Interphase Cell ◽  
Molecular Alterations ◽  
Interphase Cells

Abstract Fluorescent in situ hybridization has become a useful technique by which chromosomal abnormalities may be shown in interphase cells. We present a dual-fluorescence method whereby a chromosomal and immunophenotypic marker can be visualized simultaneously in the same interphase cell. Two patients with the myeloproliferative disorder polycythemia vera and trisomy for chromosome 8 have been studied using this technique and selective involvement of the myeloid and erythrocyte lineages has been shown by the detection of the trisomy in immunophenotyped cells. Simultaneous analysis of genotype and immunophenotype in individual cells from patients with myeloproliferative disorders or leukemia may help identify the developmental and lineage status of cells in which molecular alterations have resulted in clonal advantage.

scholarly journals A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes

1989 ◽  
Vol 86 (17) ◽  
pp. 6691-6695 ◽  
Author(s):  
B Davies ◽  
S Feo ◽  
E Heard ◽  
M Fried
Keyword(s):  
Ribosomal Protein ◽  
Recombinant Dna ◽  
Single Gene ◽  
Ribosomal Protein Gene ◽  
Pcr Primers ◽  
Pcr Product ◽  
Dna Libraries ◽  
Processed Pseudogenes ◽  
Polymerase Chain ◽  
Genomic Dna Sequence

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.

Molecular Hybridization of Mouse Satellite DNA-Complementary RNA in Ultrathin Sections Prepared for Electron Microscopy

1974 ◽  
Vol 14 (2) ◽  
pp. 253-261
Author(s):  
J. JACOB ◽  
KATHERINE GILLIES ◽  
D. MACLEOD ◽  
K. W. JONES
Keyword(s):  
Electron Microscopy ◽  
Satellite Dna ◽  
Similar Distribution ◽  
Tissue Sections ◽  
Satellite Sequences ◽  
Interphase Cells ◽  
Nucleolar Chromatin ◽  
Ultrathin Sections ◽  
Mouse Satellite Dna

The feasibility of in situ hybridization in tissue sections prepared for electron microscopy has been examined using mouse satellite DNA-complementary RNA and mouse L cells. The results obtained are encouraging, although certain technical aspects require further clarification. In interphase cells, hybrid-forming sites occur in chromatin patches positioned along the nuclear envelope. It is also confirmed that satellite DNA occurs in nucleolus-associated chromatin. The results suggest that satellite sequences are present in intranucleolar and peri-nucleolar chromatin. A similar distribution is indicated for ribosomal cistrons.

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