Human Chromosome Spreads Research Articles

FastTag Nucleic Acid Labeling System: a versatile method for incorporating haptens, fluorochromes and affinity ligands into DNA, RNA and oligonucleotides.

The FastTag Nucleic Acid Labeling System couples haptens, fluorochromes or affinity ligands to any nucleic acid by attaching a universal, photo-or heat-activatable moiety to which any sulfhydryl-reactive compound can be linked. To demonstrate the versatility of the FastTag system, we have labeled DNA, RNA and oligonucleotide probes with a variety of maleimide-coupled moieties and have used these probes in several applications. In Southern hybridization analyses, RNA probes labeled using FastTag FL (fluorescein) detected 0.04 pg of target DNA. Human satellite DNA clones labeled using FastTag FL or FastTag Biotin detected the corresponding sequences in human chromosome spreads and interphase nuclei by fluorescence in situ hybridization. An antisense oligonucleotide probe cocktail complementary to human proinsulin transcripts labeled using FastTag DNP (dinitrophenyl) detected, in situ, the appropriate transcripts in pancreatic tissue sections. Oligonucleotide primers labeled with FastTag FL were used to PCR-amplify a genomic DNA fragment, which was then detected immunologically. Finally, we discuss how DNA labeled with FastTag Fucose can be bound to a fucose-binding affinity matrix and eluted under mild conditions.

Isolation and sequence of a cDNA encoding human platelet phosphofructokinase

A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics).

The automated classification of mitotic phase for human chromosome spreads.

A system has been developed that automatically recognizes the mitotic phase of human chromosome spreads for karyotyping. Suitable spreads are classified into one of five subphases of mitosis. Classification is performed on the basis of summed chromosome length and most probable chromosome width. Classification requires 100-500 msec. A television camera scans the spread through microscope optics; computer and special purpose electronics process the video signals to generate run length histograms. The histograms are used to determine mitotic phase. Unbanded spreads, 133, were classified with a 4.5% error rate. One hundred banded spreads were classified with a 15% error rate.

Binding of quinacrine to the human Y chromosome.

Human chromosome spreads were stained with 3H-quinacrine and their fluorescence observed. The exact location of specific spreads on each slide was noted and photographs taken. Autoradiographs were then prepared so that the quinacrine fluorescence of any specific chromosome could be compared directly with the distribution of grains over the same chromosome on the autoradiograph. The Y chromosome fluoresced much more intensely than any of the other chromosomes, but there were no more grains over the Y chromosome than over the other chromosomes. Therefore the enhanced fluorescence of the human Y chromosome is not due to an increased binding of quinacrine.