The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.     

Construction and expression of mouse thymidylate synthase minigenes

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.     

Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.     

Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.

An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.     

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Molecular cloning of genomic DNA segments partially coding for human thymidylate synthase from the mouse cell transformant

Mouse cells deficient in the enzyme thymidylate synthase [TS; EC 2.1.1.45] were serially transformed with human DNA to yield primary and secondary transformants which produced human TS [Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., & Seno, T. (1983) J. Biol. Chem. 258, 48-53]. Southern blot hybridization of their genomic DNA showed that six secondary transformants examined contained in common a 5.5 kb EcoRI fragment hybridized with a human Alu sequence. From the secondary transformant genomic library constructed with phage lambda Charon 4A, two recombinant phage clones carrying Alu sequences were isolated. Restriction endonuclease mapping revealed that the insert DNAs of the two phage clones overlapped and covered a region of 19 kb in total. Within this region at least six Alu sequences were located. A 2.0 kb DNA fragment, prepared from an EcoRI fragment subcloned in plasmid pBR322 and free of Alu sequences, hybridized to a single band on RNA blots of primary and secondary transformant poly(A)+ RNA, but not to RNA of mouse wild-type and recipient cell lines. The relative amount of the presumed human TS mRNA was linearly correlated with the relative activity of human TS in various types of mouse transformant cells. These results indicate that these two phage clones contain genomic DNA sequences encoding human TS.