Susan M. Quirk

Intron mobility in the T-even phages: High frequency inheritance of group I introns promoted by intron open reading frames

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5 and the sunY endonuclease 13-15 nt 3 to the intron insertion site to generate 3-nt or 2-nt 3-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.

The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I‐TevI, a double‐strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I‐TevI, all three domains are important for formation of a cleavage‐competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I‐TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I‐TevI recognizes T‐even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.

A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4

The product of the td intron open reading frame (ORF) of phage T4 is required for high-frequency transfer of the intervening sequence from intron-plus (In+) to intron-minus (In-) alleles. In vivo studies have demonstrated that the td ORF product targets cleavage of td In- DNA, and that cleavage is correlated with intron inheritance [Quirk et al., Cell 56 (1989) 455-465]. In the present study we show by in vitro synthesis of the td intron ORF product, that the protein possesses endonuclease activity and efficiently cleaves double-stranded DNA at or near the site of intron integration. In addition, we demonstrate that intron insertion is accompanied by co-conversion of the flanking exon sequences. Co-conversion of markers within 50 nt surrounding the site of intron insertion occurred at a high frequency (80-100%), and decreased at greater distance from the intervening sequence. Co-conversion may provide a mechanism for maintaining exon-intron RNA contacts required for accurate splicing of the relocated intron. Cleavage of target DNA by an intron endonuclease and co-conversion of flanking exon sequences are both features associated with mobile introns of eukaryotes, indicating a common mechanism for intron transfer in the eukaryotic and prokaryotic kingdoms.

The regulation of uterine tissue factor by estrogen

Tissue factor (TF) is a transmembrane protein that initiates coagulation and indirectly catalyzes the conversion of prothrombin to thrombin. We previously showed that treatment of immature rats with estradiol (E2) stimulated a rapid increase in TF mRNA and protein in the uterus. Our current experiments usingin situ hybridization show that the increase in TF mRNA occurred primarily in the stromal cell layer. The effect of E2 to increase TF mRNA occurred in uterine organ cultures but not in separated epithelial and stromal cellsin vitro. Thrombin and the phorbol ester, TPA, compounds which regulate TF expression in other cell types by activation of protein kinase C (PKC), increased TF mRNA in both uterine organ cultures and in separated uteriné cells. The 5′ regulatory region of the TF gene was examined for the presence of an estrogen response element (ERE) using a plasmid, pTFCAT, containing −740 to + 15 bp of the mouse TF promoter upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. There was no response to E2 in HeLa cells cotransfected with pTFCAT and a human ER construct, pHEO. In contrast, E2 increased CAT activity in cells cotransfected with a positive-control plasmid, containing the consensus ERE cloned upstream of the thymidine kinase promoter-driven CAT gene, and pHEO. CAT activity was also increased by TPA in cells transfected with pTFCAT. In summary, E2 induces TF mRNA in uterine organ culture indicating that systemic factors are not absolutely required for the effect. However, E2 injection induces transudation of plasma prothrombin into the uterus where it may be converted to thrombin. Thus thrombin may contribute to E2-induction of TF mRNAin vivo. An ERE was not identified in the 750 bp immediately 5′ to the transcription start site of the TF gene although a TPA-responsive element was present. It is postulated that E2 may induce TF mRNA by multiple indirect pathways including stimulation of PKC and Jun and Fos transcription factors, and by generation of thrombin in the uterus.