Recombinant human immunodeficiency virus type 1 (HIV-1) integrase (IN) produced in Escherichia coli efficiently cleaves two nucleotides from the 3' end of synthetic oligonucleotide substrates which mimic the termini of HIV-1 proviral DNA. Efficient cleavage was restricted to HIV-1 substrates and did not occur with substrates derived from other retroviruses. Mutagenesis of the U5 long terminal repeat (LTR) terminus revealed only moderate effects of mutations outside the terminal four bases of the U5 LTR and highlighted the critical nature of the conserved CA dinucleotide motif shared by all retroviral termini. Integration of the endonuclease cleavage products occurs subsequent to cleavage, and evidence that the cleavage and integration reactions may be uncoupled is presented. Competition cleavage reactions demonstrated that IN-mediated processing of an LTR substrate could be inhibited by competition with LTR and non-LTR oligonucleotides. A distinguishing feature of retroviruses is integration of a DNA copy of their RNA genome into host cell DNA by a virally coded protein termed integrase (IN; 9, 12, 17, 21). Integration occurs in three stages: cleavage of two nucleotides from the 3' termini of the linear double-stranded (ds) DNA product of reverse transcription (8, 19), covalent

We have performed a functional analysis of DNA sequences upstream from the gene for IE mRNA3 of herpes simplex virus type 1. Nucleotide sequences involved in initiation and positive regulation of transcription have been defined by construction of specific deletions in vitro. Transcription was assayed in vivo by microinjection into Xenopus oocytes, or by introduction of plasmid DNA into tissue culture cells and measurement of transient expression. Three functional promoter elements have been defined: i) Sequences between -16 and -37 which are not essential for transcription but are required for accurate initiation. ii) Proximal promoter sequences which are sufficient for transcription initiation in the absence of upstream sequences. iii) Far-upstream promoter sequences (more than 108bp upstream) which increase transcription in oocytes, and contain positive regulatory sequences (-174 to -331) which respond strongly to a factor in the virus inoculum.

The locations and functions of DNA sequences involved in transcription of the gene encoding herpes simplex virus type 1 immediate early (IE) mRNAs 4 and 5 were analyzed by use of a transient-expression assay. The region upstream of the genes encoding IE mRNAs 4 and 5 was fused to the thymidine kinase gene coding sequences, and production of enzyme or RNA was measured after transfection of plasmids into BHK cells. The effect of deletions in the upstream region was determined in the absence or presence of a virus structural component which stimulates herpes simplex virus IE transcription. Two distinct units were identified. One of these was a promoter which required not more than 69 base pairs of DNA specific for the genes encoding IE mRNAs 4 and 5 upstream from the mRNA 5' terminus. The other was a far-upstream region which mediated the response to the virion component and had an upstream boundary between nucleotides -347 and -335. An origin of DNA replication was interposed between these two units. The element TAATGAGATAC , which represents a consensus sequence present in the upstream regions of all herpes simplex type 1 IE genes, appeared to be essential for stimulation by the virion component. The activity of this element was modulated by the sequences which flank it, especially by regions having extremely high contents of guanine plus cytosine and which contain a conserved unit CCCGCCC or its complement GGGCGGG .

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A cDNA library was constructed from poly(A)-rich RNA of H35 rat hepatoma cells by insertion into Lgtl 1 . Screening with an antiserum to rat liver holocytochrome-c oxidase yielded fifteen different recombinant clones. Eight clones were identified using monospecific antisera to individual subunits of the rat liver enzyme. The cDNA clones coding for subunits VIc and VIII were further characterized by DNA sequence analysis after subcloning in pUC8 and M13 mp9. The deduced amino acid sequences show 80% and 60% homology to the corresponding bovine heart subunits, respectively.From the nucleotide sequences we can conclude that subunit VIc is not synthesized as a larger precursor molecule like most other mitochondrial proteins.The size of the mRNAs coding for subunits VIc and VIII is about 450 nucleotides, as revealed by Northern blot analysis with RNAs from different tissues.The clones were further used as probes for Southern blotting. Restricted high-molecular-mass DNA showed a complex pattern of bands indicating multigene families for both subunits in the rat genome.