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A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into genes and proteins in NIH 3T3 cells. Several hundred cell clones, each expressing GFP fluorescence in a distinctive pattern, were isolated. Molecular analysis showed that a wide variety of genes and proteins, some known and some newly discovered, had been tagged. The analysis also revealed that, in the great majority of instances, the abundance and cellular location of the tagged protein mirrored that of its untagged counterpart. This approach provides a systematic means for the functional annotation of mammalian genomes and proteomes in living cells.

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Here, we describe an efficient system for epitope tagging cloned genes by CD tagging using a mini-Tn10 transposon delivery vector. The system was tested against a lambdaFIX genomic clone of the human nucleolin gene. Transfection of HeLa cells with the tagged gene led to the expression of both the appropriately spliced tagged transcript and the appropriately localized tagged protein.

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The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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The tailspike protein of Salmonella typhimurium phage P22 is a multifunctional homotrimer which is involved in the terminal reaction of phage assembly, the adsorption of the phage to susceptible cells, and the hydrolysis of the Salmonella O-antigen during the first steps of phage infection. The proteins made from 15 mutant tailspike structural genes carried on high level expression plasmids have been analyzed with respect to their in vivo stability, quaternary structure, capsid assembly activity, and enzymatic activity. Nine mutants synthesize tailspike proteins which fail to accumulate to any appreciable level in vivo, and thus these proteins are probably degraded. Four other altered proteins accumulate in vivo as soluble monomers. The remaining two altered proteins accumulate in vivo as stable trimers. Each of these two proteins is defective for at least one of the known functions of the tailspike protein. One is defective in the capsid assembly reaction and shows an unusual quaternary structural defect but is normal with respect to the enzymatic hydrolysis of O-antigen. The other is defective in the enzymatic hydrolysis of O-antigen but is normal with respect to its capsid assembly activity and quaternary structure. The known sequence changes which give rise to these altered proteins and those of previously identified mutants allow the description of possible functional and structural "domains" of this multifunctional protein.

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A simple and rapid method of creating an overlapping set of deletions in cloned DNA in preparation for sequencing has been developed. The method is based on a positive selection for Tn5 transposition into the cloned DNA fragment on a high-copy-number filamid, resolution of potential filamid dimers by filamentous phage infection, and the use of Tn5 both as a "portable" restriction enzyme site for in vitro DNA deletion and a "portable" sequencing primer binding site to initiate DNA sequencing reactions using a custom primer complementary to the outside ends of IS50. This new method has been utilized to sequence bacteriophage T4 gene 11, encoding the T4 baseplate protein gp11. The coding sequence of gene 11 is 657 bp in length, and predicts a primary structure of 219 amino acids that agrees well with the biochemical data previously obtained. DNA sequence around gene 11 suggests that the expression of genes 10, 11, and 12 of phage T4 are translationally coupled. Plasmids carrying deletions generated using this method have been used to map genetically five amber alleles of gene 11. These amber alleles were sequenced to confirm the proposed reading frame. The five amber alleles actually represent two different mutational changes at either codon 206 or 207, changing these adjacent glutamine codons to amber. The position of these amber alleles lends support to earlier studies identifying the carboxyl terminus of gp11 as essential in the interaction of P11 with baseplate protein P10 (Plishker and Berget, 1984).

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Twenty-seven new mutations in the structural gene for the Salmonella typhimurium bacteriophage P22 tailspike protein have been isolated, mapped using a powerful plasmid-based genetic system and their DNA sequence changes determined. The mutations were generated by hydroxylamine treatment of the cloned gene on a plasmid expression vector. Assaying the activity of the tailspike protein produced from this plasmid and screening for plasmid mutants were accomplished by the in situ complementation of P22 capsids imbedded in soft agar to produce infectious phage. Deletion mutations in the cloned gene have been constructed by a two step procedure involving oligonucleotide linker insertion and in vitro deletion by restriction endonuclease digestion. The deletions, whose physical endpoints were determined by DNA sequencing, define 12 genetic and physical intervals into which the new mutations were mapped by marker rescue experiments. These deletions were transferred to phage P22 by recombination and used to map mutations carried on plasmids. Following mapping, the nucleotide change for each of the mutations was determined by DNA sequencing. The majority were absolute missense mutations although both amber and ochre nonsense mutations were also identified in the protein coding portion of the gene. The suppression pattern of the nonsense mutations was determined on several nonsense suppressors. Four of the mutations cause severely depressed levels of tailspike protein expression from both the cloned gene on the plasmid expression vector and from P22 phage carrying these mutations. These mutations were identified as nucleotide changes in what is probably the P22 late operon transcription terminator which immediately follows the tailspike protein coding sequence.

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Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)

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Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.

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Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.

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The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y. Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer. During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm. Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases. Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge. P11 assembly activity is completely lost after removal of the first positive charge. These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex. Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids.

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Two bacteriophage T4 proteins which are precursors to the phage baseplate have been purified to homogeneity. These proteins, P10 and P11, are components of the P(10/11) complex, which is the first intermediate in the assembly of T4 baseplate 1/6th arms. Each protein was isolated from cells infected with a T4 amber mutant defective in the production of the other protein. Thus these purified proteins have never been assembled into the P(10/11) complex in vivo. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the ability of these proteins to block the phage killing activity of specific antisera were used to monitor the purification steps. Sedimentation equilibrium experiments reveal a molecular weight of 188,000 g/mol for P10 and 60,000 g/mol for P11. These data together with the previously determined molecular weights of the gene 10 and gene 11 polypeptide chains (King & Mykolajewycz, 1973) and the in vivo assembled P(10/11) complex (Berget & King, 1978b) are consistent with P10 being a dimer of the product of gene 10, P11 being a dimer of the product of gene 11, and P(10/11) being a tetramer containing one of each of these dimers. Purified P10 and P11 are active in assembly because they complement 10- and 11- defective extracts, respectively, to form viable bacteriophage in vitro. Furthermore, these proteins assemble in vitro to form a protein structure identical to the P(10/11) complex formed in vivo as determined by non-denaturing gel electrophoresis. This P(10/11) complex formed in vitro complements 10-/11- defective extracts to form viable phage. The overall extent of this in vitro assembly reaction is not affected by NaCl to 1.5 M or 2% Triton X-100. The reaction is, however, prevented by the denaturing effects of urea and sodium dodecyl sulfate.

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The structural gene for Salmonella bacteriophage P22 tail protein, gene 9, is separated from the remainder of the P22 late operon genes by the immI region. Early transcription of immI gene ant, which is immediately promoter proximal to gene 9, occurs in the same direction as late gene transcription but does not enter gene 9 coding sequences (Susskind & Youderian, 1982). We have cloned gene 9 and surrounding sequences into pBR322 and subsequently positioned lac UV5 promoters at varying distances before the start of gene 9 by DNA manipulations in vitro. Using an in vitro phage assembly assay to measure in vivo expression of tail protein from these plasmids and in vitro transcription reactions to measure transcriptional template activity of DNA fragments isolated from these plasmids, we have identified a region of DNA between gene ant and gene 9 that behaves as a transcription termination signal. The DNA sequence of this region shows hyphenated dyad symmetry followed by a run of seven thymine residues on the coding strand. This sequence can be drawn in a potential stem-and-loop secondary structure similar to known rho-independent transcription termination signal sequences. We discuss the role of this transcriptional terminator sequence in gene 9 expression and the early to late transcriptional switch in the P22 infection cycle.

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The tail structure of the Salmonella phage P22 mediates both adsorption of the phage to its host and enzymatic hydrolysis of the bacterial O-antigen. The tail is an oligomeric structure, which is assembled from a single polypeptide species. We report here the amino- and carboxyl-terminal sequences of the P22 tail protein and the nucleotide sequence of its gene (gene 9). These data specify the complete amino acid sequence of the tail protein. The tail protein is a slightly acidic protein containing 666 amino acids. Comparison of the gene and protein sequences indicates that mature tail protein arises by cleavage of the initiator N-formyl-methionine from the nascent chain.

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Molecular substrates for probing nonhomologous recombination in somatic cells were constructed by inserting pBR322 sequences at selected sites on the simian virus 40 (SV40) genome. The chimeric products are too large to be packaged into an SV40 capsid. Therefore, production of viable progeny requires that most of the pBR322 sequences be deleted without altering any SV40 sequences that are essential for lytic infection. As judged by plaque assay, these recombination events occur at readily detectable frequencies after transfection into CV1 monkey kidney cells. Depending on the site of pBR322 insertion, the infectivities of the full-length circular or linear chimeras ranged from 0.02 to 2% of the infectivity of linear wild-type SV40 DNA. Nucleotide sequence analysis of several recombinant progeny revealed three distinct classes of recombination junction and indicated that the causative recombination events were minimally dependent on sequence homology. Potential mechanisms involving recombination at internal sites or at ends were distinguished by measuring the infectivity of chimeric molecules from which various lengths of pBR322 had been removed. These data support end-to-end joining as the primary mechanism by which DNA segments recombine nonhomologously in somatic cells. This end joining appears to be very efficient, since SV40 genomes with complementary single-stranded tails or with short non-complementary pBR322 tails were comparably infectious. Overall, this study indicates that mammalian somatic cells are quite efficient at the willy-nilly end-to-end joining of unrelated DNA segments.

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As part of a genetic analysis of the in vivo folding and subunit assembly of the P22 tail spike endorhamnosidase, we have studied the maturation of the newly synthesized 76,000-dalton polypeptide chains into thermostable tail spike oligomers. Four of 15 temperature-sensitive mutations in the structural gene for this protein result in electrophoretically distinct tail spikes. Cells mixedly infected with wild type and an electrophoretic variant produce two hybrid species, with mobilities intermediate between the parental species, indicating that the native tail spike is a trimer. Mature trimers are resistant to denaturation by sodium dodecyl sulfate (SDS): at room temperature the trimer migrates in an SDS gel as if it were not binding significant amounts of SDS, whereas the heat-denatured chain migrates as expected of an SDS-polypeptide complex. The mature trimer is also resistant to trypsin digestion. Lysates of infected cells contain SDS and trypsin-sensitive forms of the newly synthesized tail spike polypeptide chains. These are probably incompletely or incorrectly folded chains. SDS and trypsin resistance were used to measure the efficiency of in vivo folding and subunit assembly of the mature trimer from its polypeptide chains. This decreased from 90% at 27 degrees C to only 15% at 42 degrees C. These results are consistent with the existence or a labile intermediate or step in the folding or subunit assembly of the thermostable tail spike protein. We discuss the possibility that the achievement of certain structural features of mature proteins may entail difficulties in their folding pathways.

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As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.--None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of these ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.

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The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s(20,w) of 9.3S, a D(20,w) of 4.3 x 10(-7) cm(2)/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage "tailed" with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.

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