Construction of a genomic library of the human cytomegalovirus genome and analysis of late transcription of its inverted internal repeat region

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5672144

Silva, K F.S.T.

The investigations described in this dissertation were designed to determine the transcriptionally active DNA sequences of IIR region and to identify the viral mRNA transcribed from the transcriptionally most active DNA sequences of that region during late phase of HCMV Towne infection. Preliminary transcriptional studies which included the hybridization of a southern blot of XbaI digested entire HCMV genome to {sup 32}P-labelled late phase infected cell A{sup +} RNA, indicated that late viral transcripts homologous to XbaI Q fragment of IIR region were very highly abundant while XbaI Q fragment showed a very low transcriptional activity. To facilitate further analysis of late transcription of IIR region, the entire DNA sequences of IIR region were molecularly cloned as U, S, and H BamHI fragments in pACYC-184 plasmid vector. In addition, to be used in future studies on other regions of the genome, except for y and c{prime} smaller fragments the entire 240 kb HCMV genome was cloned as BamHI fragments in the same vector. Furthermore, the U, S, and H BamHI fragments were mapped with six other restriction enzymes in order to use that mapping data in subsequent transcriptional analysis of the IIR region. Further localization of transcriptionally active DNA sequences within IIR region was achieved by hybridization of southern blots of restricted U, S, and H BamHI fragments with 3{prime} {sup 32}P-labelled infected cell late A{sup +} RNA. The 1.5 kb EcooRI subfragments of S BamHI fragment and the adjoining 0.72 kb XhoI subfragment of H BamHI fragment revealed the highest level of transcription, although the remainder of the S fragment was also transcribed at a substantial level. The U fragment and the remainder of the H fragment was transcribed at a very low level.

• Research Organization: California Univ., Davis, CA (United States)
• OSTI ID: 5672144
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550901 -- Pathology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
GENETIC MAPPING
HYBRIDIZATION
INFECTIOUS DISEASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MAN
MAPPING
MESSENGER-RNA
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
RNA
STRUCTURAL CHEMICAL ANALYSIS
TRACER TECHNIQUES
TRANSCRIPTION
VERTEBRATES
VIRAL DISEASES
VIRUSES