Structure and expression of the Chinese hamster thymidine kinase gene.

Abstract

My colleagues and I have cloned a nearly full-length Chinese hamster thymidine kinase (TK) cDNA in a AgtlO vector and characterized this cDNA by nucleotide sequencing. The hamster TK protein is encoded in this cDNA by a 702-base-pair open reading frame which specifies a 25,625-dalton protein closely homologous to the previously described human and chicken TK proteins. Using cDNA nucleotide sequence data in conjunction with sequence data derived from selected subelones of the hamster TK gene recombinant phage XHaTK.5, we have resolved the structure of the TK gene, finding the 1,219 base pairs of the cDNA sequence to be distributed through 11.2 kilobases of genomic DNA in at least seven exon segments. In addition, we have constructed a variety of Chinese hamster TK minigenes and exonuclease III-S1 derivatives of these genes which have permitted us to define the limits of the Chinese hamster TK gene promoter and demonstrate that efficient TK transformation of Ltk-cells by TK minigenes depends on the presence of both TK intervening sequences and sequences 3' to the site of mRNA polyadenylation.The enzyme thymidine kinase (TK) (EC 2.1.7.21), which salvages thymidine for DNA synthesis through an ATPdependent phosphorylation, is one of a number of enzymes important to nucleotide metabolism whose expression is cell cycle S-phase specific (for a review see Mitchison [59]). The TK activity in synchronously growing in vitro cell cultures, for example, rises sharply as cells enter and progress through S phase and then diminishes as the cells move through the G2, M, and G1 phases of the cell division cycle (10,38,39,60,72,73). As a reflection of the S phase specificity of TK gene expression, TK activity in asynchronously growing cell cultures is growth phase dependent, being maximal as cultures grow through mid-log phase and minimal as these cultures reach confluence, withdraw from the cell cycle, and accumulate in the G1/Go phase (2,16,34,37,49,50,60,64). The genetic determinants which govern this pattern of TK gene expression have not yet been precisely defined. These determinants are presumed, however, to be closely linked to TK structural gene sequences since TK gene expression in mouse Ltk-cells transformed to Tk+ with either total mouse genomic DNA or a cloned human TK gene has been shown to be S phase specific (8,68).The recent clonings of the hamster (47), human (7, 44, 48), and chicken (63) TK genes invite the possibility that the genetic determinants which underlie the cell cycle and growth phase dependence of TK gene expression can be localized within segments of these cloned TK genes through a systematic in vitro construction of TK gene mutants which can be analyzed for their pattern of expression after transfection and stable integration into a suitable Tk-host cell. My colleagues and I have previously reported the isolation and preliminary characterization of the Chinese hamster TK gene, carried in the Chinese hamster genome on chromosome 7 (70). In this report I describe (i) the molecular cloning and nucleotid...

Analysis of the Bovine Herpesvirus Type 1 Thymidine Kinase (TK) Gene from Wild-type Virus and TK-deficient Mutants

“…Moreover, recently it has been shown by means of sitedirected mutagenesis that the five highly conserved amino acids GXXGXGKT (where X represents various amino acids), which occur at residues 56 to 63 of the HSV-1 TK polypeptide, are essential for enzyme function and appear to be involved in the substrate-binding domains of the enzyme (Liu & Summers, 1988). The regions II and IV have a high degree of conservation among different herpesvirus TKs but similar sequences were not seen in poxvirus or mammalian TKs or adenylate kinases (Boyle et al, 1987 ;Bradshaw & Deininger, 1984;Kwoh & Engler, 1984;Lewis, 1986;Lin et al, 1985;Kuby et al, 1984). The functional roles of these two regions and of regions 'a' and 'b' of the polypeptide are still unknown.…”

Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle.

“…Most eucaryotic genes contain IVSs or introns that are excised from nuclear premRNA by the RNA splicing machinery (1,25,27,32). To gain insight into the nature of the putative high-molecularweight TK pre-mRNAs present in G,/S-and S-phase nuclei, as well as to determine whether there is a preferential order of IVS removal from the TK gene, we further characterized the TK pre-mRNA splicing intermediates present in S-phase cells by Northern (RNA) blot analysis.Mammalian TK genes, which contain seven coding exons and six introns, have been well conserved during evolution (6,20,23,31). We isolated unique sequence DNA probes from each of the six TK introns and used them to character-* Corresponding author.…”

“…Mammalian TK genes, which contain seven coding exons and six introns, have been well conserved during evolution (6,20,23,31). We isolated unique sequence DNA probes from each of the six TK introns and used them to character-* Corresponding author.…”

“…Therefore, many different factors, including sequences at splice junctions (18,19) and lengths and sequences of introns (7,29) as well as sequences of adjacent exons (28,30), probably contribute unique secondary structural features to premRNAs which favor the interactions of specific spliceosome complexes over those of others. Many characteristics, including the overall intron-exon organization and the nucleotide sequences located at individual splice junctions, have been conserved in mammalian TK genes (6,20). The observation that the same ordered pattern of intron removal has been preserved in mice, in Chinese hamsters, and probably in humans (J. M. Gudas, unpublished results) can most likely be attributed to secondary and tertiary structural features of nascent TK transcripts which favor the interactions of specific spliceosome complexes over those of others.…”

Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells.

“…These results suggested that passage through a splicing pathway might be a general requirement for formation of stable cytoplasmic mRNA. Such a requirement could explain the poor transformation efficiency of various intronless minigenes (5, 16,17). However, rigorous reaffirmation of the importance of introns to eucaryotic mRNA formation has not been reported.…”