Amplification of a transcriptionally active DNA sequence in the human brain

Journal Article · Sat Mar 01 04:00:00 EST 1986 · Dokl. Biol. Sci. (Engl. Transl.); (United States)

OSTI ID:5856095

Yakovlev, A G; Sazonov, A E; Spunde, A Ya; Gindilis, V M

The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A/sup +/) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A/sup +/) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10/sup 5/ recombinant. This library was analyzed by the hybridization method on columns with two radioactive (/sup 32/P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with (/sup 32/P) to a specific activity of 0.5-1 x 10/sup 9/ counts/min x microgram. The autograph was exposed with amplifying screens at -70/sup 0/C for 2 days.

• Research Organization: All-Union Scientific Center for Mental Health, Moscow, USSR
• OSTI ID: 5856095
• Journal Information: Dokl. Biol. Sci. (Engl. Transl.); (United States), Journal Name: Dokl. Biol. Sci. (Engl. Transl.); (United States) Vol. 284:1-6; ISSN DKBSA
• Country of Publication: United States
• Language: English

Related Subjects

550401* -- Genetics-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
BODY
BRAIN
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CHROMATOGRAPHY
CLONING
DAYS LIVING RADIOISOTOPES
DNA
DNA REPLICATION
DNA SEQUENCING
DNA-CLONING
GENES
HYBRIDIZATION
ISOTOPES
LABELLED COMPOUNDS
LABELLING
LIGHT NUCLEI
LIVER CELLS
MAMMALS
MAN
NERVE CELLS
NERVOUS SYSTEM
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PLASMIDS
PRIMATES
RADIOASSAY
RADIOISOTOPES
RATS
RECOMBINANT DNA
RNA
RODENTS
SEPARATION PROCESSES
SOMATIC CELLS
STRUCTURAL CHEMICAL ANALYSIS
SYNTHESIS
TRANSCRIPTION
VERTEBRATES