Host-specificities of papillomavirus, Moloney murine sarcoma virus and simian virus 40 enhancer sequences.

The bovine papillomavirus (BPV-1), Moloney murine sarcoma virus (MoMuSV) and simian virus 40(SV40) genomes have been shown to contain sequences termed 'enhancers' which activate the expression of linked genes. DNA fragments containing these three enhancers have been inserted into recombinant plasmids upstream from the herpes simplex virus thymidine kinase (tk) gene, and their effect on tk expression monitored. Two types of assay have been used. Firstly, the ability of recombinant plasmids to transform TK- recipient cells to a TK+ phenotype was measured. Secondly, the amount of tk-specific RNA and TK enzyme activity transiently expressed after DNA transfection was determined. Both types of assay gave similar results. The enhancers increased tk gene expression by regulating the amount of full length tk mRNA present shortly after transfection independent of gene copy number. Furthermore, marked species specificity in the relative efficiencies of different enhancers was observed, including that of the BPV-1 enhancer for the first time. The MoMuSV enhancer showed preference for murine fibroblasts, while the papillomavirus enhancer showed a marked preference for bovine cells. In contrast, the SV40 enhancer gave the same relative increase in tk gene expression in the murine, rat, bovine and human cells tested.     

Chicken ovalbumin gene fused to a herpes simplex virus alpha promoter and linked to a thymidine kinase gene is regulated like a viral gene.

We are describing a system for the introduction, selection, and expression of eucaryotic genes in higher eucaryotic cells. The carrier consisted of the herpes simplex virus 1 (HSV-1) tk gene covalently linked to an HSV-1 alpha promoter directed away from the tk gene. In this study we fused to the alpha promoter the 5' transcribed noncoding sequences and the coding sequences of the chicken oviduct ovalbumin gene. Cells converted to the TK+ phenotype with this chimeric fragment produced an ovalbumin precursor which was processed and secreted into the extracellular fluid. The ovalbumin gene utilized the HSV-1 alpha promoter and was regulated as a viral gene inasmuch as inversion of the genomic DNA relative to the alpha promoter resulted in no ovalbumin synthesis, and production of ovalbumin was enhanced after superinfection with HSV-1. Synthesis of ovalbumin was not detected when cDNA was linked to the HSV-1 alpha promoter. The carrier system described in this study is suitable for introduction, selection, and expression of eucaryotic genes whose natural promoter is either weak or requires the presence of regulatory elements which may be absent from undifferentiated cells in culture.     

Independent regulation of thymidine kinase mRNA and enzyme levels in serum-stimulated cells.

We have studied the regulation of thymidine kinase mRNA and protein/enzyme expression in quiescent and serum-stimulated rat cells transfected with a human TK cDNA clone expressed from a number of promoters. Our results indicate that while the pattern of mRNA expression is a function of the promoter used, the pattern of protein/enzyme expression is not. When the gene is expressed from the homologous human TK promoter both mRNA and enzyme levels remain low throughout G1 and increase as the cells enter S phase. When it is expressed from the heterologous SV40 early promoter, mRNA levels are high throughout G1, but enzyme and protein levels remain low until 8-10 h following serum stimulation. Thus, protein levels appear to be uncoupled from mRNA levels in this system, suggesting the presence of translational and/or posttranslational regulation. An analysis of mutant cDNA clones indicates that this regulation is not dependent upon sequences at the 5' or 3' end of the cDNA, including the entire 5'-untranslated region, the authentic AUG and the first 48 nucleotides of the coding region.     

Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences

As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of TK promoter-chloramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -174 base pairs upstream and +159 base pairs downstream of the TK translation initiation site that are sufficient to drive efficient S phase-specific expression of the CAT reporter gene in transfected murine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Sp1 consensus binding site is critical to the activity of this promoter. Synchronized populations of BALB/c 3T3 cells stably transfected with either TK promoter-CAT fusion constructs or TK promoter-beta-globin fusion constructs expressed their respective reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these constructs therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans-acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.     

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.     

Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.

To study the influence of clustered highly repetitive DNA sequences on the expression of adjacent genes, LTK- cells were cotransfected with the herpes simplex virus thymidine kinase (tk) gene and mouse satellite DNA. TK+ transformants containing a few copies of the tk genes flanked by satellite DNA were isolated. In situ hybridization on the metaphase chromosomes indicated that in each cell line the TK sequences resided at a single chromosomal site and that integration occurred preferentially into regions of the cellular DNA rich in highly repetitive sequences. The prominent feature of these cell lines was their phenotypic instability. Suppression and reexpression of the tk gene occurred at high frequency (greater than 3%) and did not correlate with any significant change in the organization of foreign DNA or with the presence of selective agents. These results indicate that satellite DNA, the major component of constitutive heterochromatin, may influence the expression of adjacent genes by affecting the chromatin structure.     

Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene.

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.     

Activation of an enhancerless gene by chromosomal integration.

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.