The td, ttrdB, and sunY introns of bacteriophage T4 each contain a long open reading frame (ORF). These ORFs are preceded by functional T4 late promoters and, in the case of the nrdB intron ORF, a functional middle promoter. Expression of phage-encoded intron ORF-IacZ fusions indicates that these T4 genes are highly regulated. The lack of translation of these ORFs from the early pre-mRNAs can be accounted for by the presence of secondary structures that are absent from the late RNAs. Because translation of the intron ORFs could disrupt core structural elements required for pre-mRNA splicing, such regulation may be necessary to allow expression of the genes in which they reside.

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The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 anmo acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.The T4 sun Y gene is one of four bacteriophage genes that have been identified as containing self-splicing group I introns (7,12,30,32 deletion phages SaA9 and rNB2226 were provided by L. Gold. SaA9 contains a 2.4-kb deletion in the D region extending from ac through Dl (9), while rNB2226 contains an approximately 6-kb deletion spanning the rII region (1). E. coli XA101 [F-ara A(lac-pro) nalA supD metB argE(Am) rifthi] was from E. Eisenstadt. T4 alt (ac-q) (15) phage were from C. Goff, and amber mutants in T4 genes 21 (amHll) and 23 (amE322-amN12l) were from the collection of L. Black. J. Karam provided T4 gene 55 amber mutant amBL292 (3). T4 deletions farPl3, farP23, tk2, and tk25 (6) were from D. Hall, and deletion A9A (21), isolated by D.Parma, was from J. Wiberg.Descriptions of all relevant plasmids are given in intron (see Fig. 1). The rIIB-lacZ gene fusion of pAHZ2 was transferred to the sun Y gene of T4 rNB2226 by homologous recombination during infection of E. coli XA101 harboring the plasmid. The new phage was designated rNB2226, sun Y[rIIBlacAHZ2].A deletion within the sun Y gene was created in plasmid pAAX3 (Table 1) The sun Y deletion was introduced into phage, with or without a gene 55 amber mutation (amBL292), by recombination between plasmid DNA bearing the sun Y deletion and homologous sequences in the phage. The recipient phage used in this experiment contained a fusion of the sun Y intron open reading frame (ORF) with lacZ (13). Recombinant phage (containing the deletion) were identified by loss of P-galactosidase and were designated sun YA3 or 55am, sunYA3.Transfer of plasmid sequences to T4. Plasmid-containing strains of E. coli were grown in M9S medium (3) supplemented with 50 ,ug of ampicillin per ml to a density of 2 x 10R cells per ml and infected at a multiplicity of 1. After 5 min at 30°C, the cultures were diluted 100-fold into M9S medium and incubated for 1 to 2 h. Chloroform was added to lyse cells remaining in the culture. The progeny were plated and screened for their ability to form blue plaques on plates containing 0.04% 5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside (X-Gal) with a lac deletion lawn (E. coli XA101) (or for the loss of the ability to form blue plaques in the case of the sun Y internal del...

Teachers are being challenged to engage students in ways that will elevate student interest and understanding of concepts in science and encourage students to gather evidence to support what we know about science. It is critical for teachers to have budget-friendly, supporting activities that are aligned with current educational standards, that are easy to present, and that incorporate a variety of skills important to the study of science. This simulation activity is an inexpensive exercise that challenges students to hypothesize the evolutionary outcome of genotype selection in a population and then gather and analyze a set of unique data. Connections to recent evolutionary changes in familiar organisms ground the activity for students, helping them grasp the importance of such investigative work.